Treatment of addiction and addiction-related behavior

ABSTRACT

The present invention provides a method for changing addiction-related behavior of a mammal suffering from addiction to a combination of abused drugs. The method includes administering to the mammal an effective amount of gamma vinylGABA (GVG) or a pharmaceutically acceptable salt thereof, or an enantiomer or a racemic mixture thereof, wherein the effective amount is sufficient to diminish, inhibit or eliminate behavior associated with craving or use of the combination of abused drugs.

The present application is a continuation-in-part of co-pendingapplication Ser. No. 09/209,952 filed Dec. 11, 1998, which is acontinuation in part of application Ser. No. 09/189,166 filed Nov. 9,1998, which is a continuation-in-part of application Ser. No. 09,129,253filed on Aug. 5, 1998, now U.S. Pat. No. 6,057,368 issued May 2, 2000.

This invention was made with Government support under contract numberDE-AC02-98CH10886, awarded by the U.S. Department of Energy. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to the use of an irreversible inhibitor ofGABA-transaminase for the treatment of substance addiction andmodification of behavior associated with substance addiction. Substanceaddiction, such as drug abuse, and the resulting addiction-relatedbehavior are enormous social and economic problems that continue to growwith devastating consequences.

Substance addiction can occur by use of legal and illegal substances.Nicotine, cocaine, amphetamine, methamphetamine, ethanol, heroin,morphine and other addictive substances are readily available androutinely used by large segments of the United States population.

Many drugs of abuse are naturally occurring. For example, cocaine is anaturally occurring nonamphetamine stimulant derived from the leaves ofthe coca plant, Erythroylon coca. Coca leaves contain only aboutone-half of one percent pure cocaine alkaloid. When chewed, onlyrelatively modest amounts of cocaine are liberated, and gastrointestinalabsorption is slow. Certainly, this explains why the practice of chewingcoca leaves has never been a public health problem in Latin America. Thesituation changes sharply with the abuse of the alkaloid itself.

It has been found that addicting drugs such as nicotine, cocaine,amphetamine, methamphetamine, ethanol, heroin, and morphine enhance (insome cases directly, in other cases indirectly or eventrans-synaptically) dopamine (DA) within the mesotelencephalicreward/reinforcement circuitry of the forebrain, presumably producingthe enhanced brain reward that constitutes the drug user's “high.”Alterations in the function of these DA systems have also beenimplicated in drug craving and in relapse to the drug-taking habit inrecovering addicts. For example, cocaine acts on these DA systems bybinding to the dopamine transporter (DAT) and preventing DA reuptakeinto the presynaptic terminal.

There is considerable evidence that nicotine, cocaine, amphetamine,methamphetamine, ethanol, heroin, morphine and other abused drugs'addictive liability is linked to reuptake blockade in central nervoussystem (CNS) reward/reinforcement pathways. For example, cocaine-inducedincreases in extracellular DA have been linked to its rewarding andcraving effects in rodents. In humans, the pharmacokinetics bindingprofile of ¹¹C-cocaine indicates that the uptake of labeled cocaine isdirectly correlated with the self-reported “high”. In addition, humancocaine addicts exposed to cocaine-associated environmental cuesexperienced increased cocaine craving which is antagonized by the DAreceptor antagonist haloperidol. Based upon the presumptive link betweencocaine's addictive liability and the DA reward/reinforcement circuitryof the forebrain, many pharmacologic strategies for treating cocaineaddiction have been proposed.

In the past, one treatment strategy was to target directly the DAT witha high-affinity cocaine analog, thereby blocking cocaine's binding.Another treatment strategy was to modulate synaptic DA directly by theuse of DA agonists or antagonists. Yet another treatment strategy was tomodulate synaptic DA, indirectly or trans-synaptically, by specificallytargeting a functionally-linked but biochemically differentneurotransmitter system.

A number of drugs have been suggested for use in weaning cocaine usersfrom their dependency. Certain therapeutic agents were favored by the“dopamine depletion hypothesis.” It is well established that cocaineblocks dopamine reuptake, acutely increasing synaptic dopamineconcentrations. However, in the presence of cocaine, synaptic dopamineis metabolized as 3-methoxytyramine and excreted. The synaptic loss ofdopamine places demands on the body for increased dopamine synthesis, asevidenced by the increase in tyrosine hydroxylase activity after cocaineadministration. When the precursor supplies are exhausted, a dopaminedeficiency develops. This hypothesis led to the testing ofbromocriptine, a dopamine receptor agonist. Another approach was theadministration of amantadine, a dopamine releaser. Yet another approach,also based on the dopamine depletion hypothesis, was to provide aprecursor for dopamine, such as L-dopa.

Agonists are not preferred therapeutic agents. A given agonist may acton several receptors, or similar receptors on different cells, not juston the particular receptor or cell one desires to stimulate. Astolerance to a drug develops (through changes in the number of receptorsand their affinity for the drug), tolerance to the agonist may likewisedevelop. A particular problem with the agonist bromocriptine, forexample, is that it may itself create a drug dependency. Thus, treatmentstrategies used in the past did not relieve the patient's craving forcocaine. Moreover, by using certain agonists such as bromocriptine, apatient was likely to replace one craving for another.

Another drug that is frequently abused is nicotine. The alkaloid(−)-nicotine is present in cigarettes and other tobacco products thatare smoked or chewed. It has been found that nicotine contributes tovarious diseases, including cancer, heart disease, respiratory diseaseand other conditions, for which tobacco use is a risk factor,particularly heart disease.

Vigorous campaigns against the use of tobacco or nicotine have takenplace, and it is now common knowledge that the cessation of tobacco usebrings with it numerous unpleasant withdrawal symptoms, which includeirritability, anxiety, restlessness, lack of concentration,lightheadedness, insomnia, tremor, increased hunger and weight gain,and, of course, an intense craving for tobacco.

The addictive liability of nicotine has been linked to therewarding/reinforcing actions and its effects on DA neurons in thereward pathways of the brain (Nisell et al., 1995; Pontieri, et al.,1996). For example, the acute systemic administration of nicotine, aswell as numerous other drugs of abuse, produces an increase inextracellular DA levels in the nucleus accumbens (NACC), an importantcomponent of the reward system (Damsma et al., 1989; Di Chiara andImperato, 1988; Imperato et al., 1986; Nisell et al., 1994a, 1995;Pontieri et al., 1996). Similarly, the infusion of nicotine into theventral segmental area (VTA) of the rodent produces a significantincrease in DA levels in the NACC (Nisell et al., 1994b).

A few pharmaceutical agents have been reported as useful to treatnicotine dependence, including nicotine substitution therapy such asnicotine gum, transdermal nicotine patches, nasal sprays, nicotineinhalers and bupropion, the first nonnicotinic treatment for smokingcessation (Henningfield, 1995; Hurt, et al., 1997).

Unfortunately, nicotine substitution therapy involves the administrationof the nicotine which frequently leads to nicotine withdrawal andsubsequent relapse to use of tobacco products. Thus, there is a need fora therapy having a desirable side effect profile, to relieve nicotinewithdrawal symptoms, including the long term cravings for nicotine.

Other well known addictive substances are narcotic analgesics such asmorphine, heroin and other opioids both natural and semisynthetic. Abuseof opioids induce tolerance and dependence. Withdrawal symptoms from thecessation of opioids use vary greatly in intensity depending on numerousfactors including the dose of the opioid used, the degree to which theopioid effects on the CNS are continuously exerted, the duration ofchronic use, and the rate at which the opioid is removed from thereceptors. These withdrawal symptoms include craving, anxiety,dysphoria, yawning, perspiration, lacrimation, rhinorrhoea, restless andbroken sleep, irritability, dilated pupils, aching of bones, back andmuscles, piloerection, hot and cold flashes, nausea, vomiting, diarrhea,weight loss, fever, increased blood pressure, pulse and respiratoryrate, twitching of muscles and kicking movements of the lowerextremities.

Medical complications associated with injection of opioids include avariety of pathological changes in the CNS including degenerativechanges in globus pallidus, necrosis of spinal gray matter, transversemyelitis, amblyopia, plexitis, peripheral neuropathy, Parkinsoniansyndromes, intellectual impairment, personality changes, andpathological changes in muscles and peripheral nerves. Infections ofskin and systemic organs are also quite common including staphylococcalpneumonitis, tuberculosis, endocarditis, septicemia, viral hepatitis,human immunodeficiency virus (HIV), malaria, tetanus and osteomyelitis.The life expectancy of opioid addicts is markedly reduced, due tooverdose, drug-related infections, suicide and homicide.

Pharmaceutical agents used in treating opioid dependence includemethadone, which is an opioid, and opioid antagonists, primarilynaloxone and naltrexone. Clonidine has been shown to suppress someelements of opioid withdrawal but suffers from the side effects ofhypotension and sedation, which can be quite extreme. Behavior-modifyingpsychological treatment and training are frequently adjunctive therapyused in association with pharmaceutical agents. There is a need for atherapy having a more desirable side effect profile, to relieve opioidaddiction and withdrawal symptoms.

Ethanol is probably the most frequently used and abused depressant inmost cultures and a major cause of morbidity and mortality. Repeatedintake of large amounts of ethanol can affect nearly every organ systemin the body, particularly the gastrointestinal tract, cardiovascularsystem, and the central and peripheral nervous systems. Gastrointestinaleffects include gastritis, stomach ulcers, duodenal ulcers, livercirrhosis, and pancreatitis. Further, there is an increased rate ofcancer of the esophagus, stomach and other parts of the gastrointestinaltract. Cardiovascular effects include hypertension, cardiomyopathy andother myopathies, significantly elevated levels of triglycerides andlow-density lipoprotein cholesterol. These cardiovascular effectscontribute to a marked increase risk of heart disease. Peripheralneuropathy may be present as evidenced by muscular weakness,parathesias, and decreased peripheral sensation. Central nervous systemeffects include cognitive deficits, severe memory impairmentdegenerative changes in the cerebellum, and ethanol-induced persistingamnesiac disorder in which the ability to encode new memory is severelyimpaired. Generally, these effects are related to vitamin deficiencies,particularly the B vitamins.

Individuals with ethanol dependence or addiction exhibit symptoms andphysical changes including dyspepsia, nausea, bloating, esophagealvarices, hemorrhoids, tremor, unsteady gait, insomnia, erectiledysfunction, decreased testicular size, feminizing effects associatedwith reduced testosterone levels, spontaneous abortion, and fetalalcohol syndrome. Symptoms associated with ethanol cessation orwithdrawal include nausea, vomiting, gastritis, hematemises, dry mouth,puffy blotchy complexion, and peripheral edema.

The generally accepted treatment of ethanol addiction and withdrawal isaccomplished by administering a mild tranquilizer such achlordiazepoxide. Typically, vitamins, particularly the B vitamins, arealso administered. Optionally, magnesium sulfate and/or glucose are alsoadministered. Nausea, vomiting and diarrhea are treated symptomaticallyat the discretion of the attending physician. Disulfiram may also beadministered for help in maintaining abstinence. If ethanol is consumedwhile on disulfiram, acetaldehyde accumulates producing nausea andhypotension. There is a need for a therapy having a more desirable sideeffect profile, to relieve ethanol addiction and withdrawal symptoms.

Recently, it has been reported that polydrug or combination drug abusehas been increasing at an alarming rate. For example, cocaine and heroinare often abused together in a drug combination known as a“speedballing.” Such reported increase is believed to be a result of asynergistic effect that increases the euphoria of the user.

Accordingly, there is still a need in the treatment of addiction todrugs of abuse to provide new methods which can relieve a patient'scraving by changing the pharmacological actions of drugs of abuse in thecentral nervous system. There is also a need to provide new methods totreat combination drug abuse.

SUMMARY OF THE PRESENT INVENTION

The present invention, which addresses the needs of the prior art,provides methods for treating substance addiction and changingaddiction-related behavior of a mammal, for example a primate, sufferingfrom substance addiction by administering to the mammal an effectiveamount of a pharmaceutical composition including gamma vinylGABA (GVG).The amount of GVG varies from about 15 mg/kg to about 2 gm/kg,preferably from about 100 mg/kg to about 600 mg/kg, and most preferablyfrom about 150 mg/kg to about 300 mg/kg.

In a preferred embodiment, the present invention provides a method ofeliminating the effects of nicotine addiction by treating a mammal withan effective amount of a composition including GVG. When treating theeffects of nicotine addiction the amount of GVG present in thepharmaceutical composition is from about 15 mg/kg to about 2 g/kg.Preferably, 75 mg/kg to about 150 mg/kg, and most preferably from about18 mg/kg to about 20 mg/kg.

In yet another embodiment, the present invention provides a method forchanging addiction-related behavior of a mammal suffering from addictionto drugs of abuse which comprises administering to the mammal aneffective amount of GVG or a pharmaceutically acceptable salt thereof,wherein the effective amount attenuates the rewarding/incentive effectsof drugs of abuse selected from the group consisting ofpsychostimulants, narcotic analgesics, alcohols, nicotine andcombinations thereof in the absence of altering rewarding/incentiveeffects of food in said mammal.

The amount of GVG varies from about 15 mg/kg to about 2 gm/kg,preferably from about 15 mg/kg to about 600 mg/kg, and most preferablyfrom about 150 mg to about 600 mg/kg.

As a result of the present invention, methods of reducing substanceaddiction and changing addiction-related behavior are provided which arebased on a pharmaceutical composition which is not itself addictive, yetis highly effective in ameliorating the addiction and the addictivebehavior of addicted patients. The pharmaceutical composition useful forthe method of the present invention inhibits or eliminates cravingexperienced by drug addicts for use of the drug of abuse. Moreover, theelimination of behavior associated with drugs of abuse occurs in theabsence of an aversive or appetitive response to GVG. Moreover, behaviorcharacteristics associated with dependency on drugs of abuse are reducedor eliminated in the absence of an alteration in the locomotor functionof the primate.

In yet another embodiment, the invention includes a method for changingaddiction-related behavior of a mammal suffering from addiction to drugsof abuse which comprises administering to the mammal an effective amountof GVG or a pharmaceutically acceptable salt thereof, or an enantiomeror a racemic mixture thereof, wherein the effective amount is sufficientto diminish, inhibit or eliminate behavior associated with craving oruse of drugs of abuse.

In another exemplary embodiment of the present invention, the methodincludes changing addiction-related behavior of a mammal suffering fromaddiction to drugs of abuse which comprises administering to the mammalan effective amount of a composition that increases central nervoussystem GABA levels wherein the effective amount is sufficient todiminish, inhibit or eliminate behavior associated with craving or useof drugs of abuse.

In yet another exemplary embodiment, the present invention provides amethod for changing addiction-related behavior of a mammal sufferingfrom addiction to a combination of abused drugs which comprisesadministering to the mammal an effective amount of GVG or apharmaceutically acceptable salt thereof, or an enantiomer or a racemicmixture thereof, wherein the effective amount is sufficient to diminish,inhibit or eliminate behavior associated with craving or use of thecombination of abused drugs.

Other improvements which the present invention provides over the priorart will be identified as a result of the following description whichsets forth the preferred embodiments of the present invention. Thedescription is not in any way intended to limit the scope of the presentinvention, but rather only to provide a working example of the presentpreferred embodiments. The scope of the present invention will bepointed out in the appended claims.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a graph illustrating percent change in distribution volume(DV) for three groups of animals treated with cocaine.

FIG. 2 is a photograph of transaxial parametric DV ratio images of thenon-human primate brain at the level of the corpus striatum.

FIGS. 3A and 3B are graphs illustrating the effects of GVG on locomotorbehavior as compared with saline controls.

FIG. 4 is a graph illustrating the effects of GVG on nicotine-inducedextracellular dopamine.

FIGS. 5A and 5B are graphs illustrating the effects of nicotine and GVGon extracellular dopamine levels in the nucleus accumbens of freelymoving rats.

FIG. 6 is a graph illustrating the effects of methamphetamine onextracellular dopamine levels in the nucleus accumbens of freely movingrats.

FIG. 7 is a graph illustrating the effects of GVG on methamphetamineinduced changes in extracellular dopamine levels in the nucleusaccumbens of freely moving rats.

FIG. 8 is a graph illustrating the effects of GVG on alcohol inducedchanges in extracellular dopamine levels in the nucleus accumbens offreely moving rats.

FIG. 9 is a graph illustrating the effects of GVG on cocaine, heroin,and the combination of cocaine and heroin on extracellular dopaminelevels in the nucleus accumbens of freely moving rats.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a highly efficient method for treatingsubstance addiction and for changing addiction-related behavior ofmammals, for example primates, suffering from substance addiction.

As used herein, addiction-related behavior means behavior resulting fromcompulsive substance use and is characterized by apparent totaldependency on the substance. Symptomatic of the behavior is (I)overwhelming involvement with the use of the drug, (ii) the securing ofits supply, and (iii) a high probability of relapse after withdrawal.

For example, a cocaine user experiences three stages of drug effects.The first, acute intoxication (“binge”), is euphoric, marked bydecreased anxiety, enhanced self-confidence and sexual appetite, and maybe marred by sexual indiscretions, irresponsible spending, and accidentsattributable to reckless behavior. The second stage, the (“crash”),replaces euphoria by anxiety, fatigue, irritability and depression. Someusers have committed suicide during this period. Finally, the thirdstage, “anhedonia,” is a time of limited ability to derive pleasure fromnormal activities and of craving for the euphoric effects of cocainewhich leads to use of this drug. See Gawin and Kleber, MedicalManagement of Cocaine Withdrawal, 6-8 (APT Foundation). As related tococaine users, addiction-related behavior includes behavior associatedwith all three stages of drug effects.

Abused Drugs

Drugs of abuse include but are not limited to psychostimulants, narcoticanalgesics, alcohols and addictive alkaloids such as nicotine orcombinations thereof. Some examples of psychostimulants include but arenot limited to amphetamine, dextroamphetamine, methamphetamine,phenmetrazine, diethylpropion, methylphenidate, cocaine andpharmaceutically acceptable salts thereof.

Specific examples of narcotic analgesics include alfentanyl,alphaprodine, anileridine, bezitramide, codeine, dihydrocodeine,diphenoxylate, ethylmorphine, fentanyl, heroin, hydrocodone,hydromorphone, isomethadone, levomethorphan, levorphanol, metazocine,methadone, metopon, morphine, opium extracts, opium fluid extracts,powdered opium, granulated opium, raw opium, tincture of opium,oxycodone, oxymorphone, pethidine, phenazocine, piminodine,racemethorphan, racemorphan, thebaine and pharmaceutically acceptablesalts thereof.

Drugs of abuse also include CNS depressants such as barbiturates,chlordiazepoxide, and alcohols such as ethanol, methanol and isopropylalcohol.

The method of the present invention can be used to treat mammalsaddicted to a combination of drugs of abuse. For example, the mammal maybe addicted to ethanol and cocaine, in which case the present inventionis particularly suited for changing addiction-related behavior of themammal by administering an effective amount of GVG.

As used herein combination of abused drugs include combinations ofpsychostimulants, narcotic analgesics, alcohols and addictive alkaloidsas discussed above. For example, combinations of abused drugs includecocaine, nicotine, methamphetamine, ethanol, morphine and heroin. Ahighly abused combination is cocaine and heroin.

There is synergy observed with use of a combination of abused drugs. Forexample, when heroin, an indirect dopamine releaser and cocaine, adopamine reuptake inhibitor, are administered to rodents, a synergisticincrease is observed in cerebral NAc dopamine levels. Synergy may beshown, for example, by greater increases in cerebral dopamine levelsthan would be expected with either drug alone. Preferably, synergy isdemonstrated by from about 500% to about 1000% increase in cerebral NAcdopamine levels with the combination of cocaine and heroin as comparedto administering either drug alone.

Compulsive drug use includes three independent components: tolerance,psychological dependence and physical dependence. Tolerance produces aneed to increase the dose of the drug after several administration inorder to achieve the same magnitude of effect. Physical dependence is anadaptive state produced by repeated drug administration and whichmanifests itself by intense physical disturbance when drugadministration is halted. Psychological dependence is a conditioncharacterized by an intense drive, craving or use for a drug whoseeffects the user feels are necessary for a sense of well being. SeeFeldman, R. S. and Quenzer, L. F. “Fundamentals ofNeuropsychopharmocology” 418-422 (Sinaur Associates, Inc.) 1984incorporated herein by reference as if set forth in full. Based on theforegoing definitions, as used herein “dependency characteristics”include all characteristics associated with compulsive drug use,characteristics that can be affected by biochemical composition of thehost, physical and psychological properties of the host.

As explained above, the compulsive use of drugs of abuse or to thecombination of abused drugs gives rise to a euphoric stage followed by astage of craving for the euphoric effects of that drug which leads touse of the drug or combination of drugs. As used herein therewarding/incentive effects of drugs of abuse refers to any stimulus (inthis case, a drug) that produces anhedonia or increases the probabilityof a learned response. This is synonymous with reinforcement. Withrespect to experimental animals, a stimulus is deemed to be rewarding byusing paradigms that are believed to measure reward. This can beaccomplished by measuring whether stimuli produce an approach response,also known as an appetitive response or a withdrawal response, as whenthe animal avoids the stimuli, also known as an aversive response.Conditioned place preference (CPP) is a paradigm which measures approach(appetitive) or withdrawal (aversive) responses. One can infer thatrewarding stimuli produce approach behavior. In fact, one definition ofreward is any stimulus that elicits approach behavior. Furthermore, theconsequences of reward would be to enhance the incentive properties ofstimuli associated with the reward.

Reward can also be measured by determining whether the delivery of areward is contingent upon a particular response, thereby increasing theprobability that the response will reappear in a similar situation, i.e.reinforcement paradigm. For example, a rat pressing a bar a certainnumber of times for an injection of cocaine is an example ofreinforcement. Yet another way to measure reward is by determining if astimulus (e.g. a drug), through multiple pairings with neutralenvironmental stimuli, can cause the previously neutral environmentalstimuli to elicit behavioral effects initially only associated with thedrug—this conditioned reinforcement. CPP is considered to be a form ofconditioned reinforcement.

The incentive motivational value of a drug (or other stimuli) can beassessed using conditioned place preference (CPP). With respect tococaine, nicotine, heroin, morphine methamphetamine, ethanol or otherdrugs of abuse or combinations thereof, animals are tested in adrug-free state, to determine whether they prefer an environment inwhich they previously received the abused drug as compared to anenvironment in which they previously received saline. In the CPPparadigm, animals are given a drug in one distinct environment and aregiven the appropriate vehicle in an alternative environment. The CPPparadigm is widely used to evaluate the incentive motivational effectsof drugs in laboratory animals (Van Der Kooy, 1995). Followingconditioning or pairing with the drug if the animal, in a drug-freestate, consistently chooses the environment previously associated withthe drug of abuse, the inference is drawn that the appetitive value ofthe drug of abuse was encoded in the brain and is accessible in thedrug-free state. CPP is reflected in an increased duration spent in thepresence of the drug-associated stimuli relative to vehicle-injectedcontrol animals. It can also be used to asses addiction to a combinationof abused drugs.

It has been postulated that since craving at the human level is oftenelicited by sensory stimuli previously associated with drug-taking,conditioning paradigms like CPP may be used to model craving inlaboratory animals.

As used herein, craving an abused drug or a combination of abused drugsis an intense desire to self-administer the drug(s) previously used bythe mammal. The mammal does not need the abused drug to preventwithdrawal symptoms.

The addictive liability of drugs of abuse, such as for example, cocaine,nicotine, methamphetamine, morphine, heroin, ethanol or other drugs ofabuse has been linked to their pharmacological actions onmesotelencephalic dopamine (DA) reinforcement/reward pathways in thecentral nervous system (CNS). Dopaminergic transmission within thesepathways is modulated by gamma-amino butyric acid (GABA).

For example cocaine, nicotine, methamphetamine, morphine, heroin andethanol inhibit the presynaptic reuptake of monoamines. Dopaminergicneurons of the mesocorticolimbic DA system, whose cell bodies lie withinthe ventral tegmental area (VTA) and project primarily to the nucleusaccumbens (NACC), appear to be involved in cocaine, nicotine,methamphetamine, morphine, heroin or ethanol reinforcement. Electricalstimulation of reward centers within the VTA increases extracellular DAlevels in the NACC, while 6-hydroxy dopamine lesions of the NACC abolishcocaine, nicotine, methamphetamine, morphine, heroin or ethanolself-administration. In vivo microdialysis studies confirm cocaine,nicotine, methamphetamine, morphine, heroin and ethanol's ability toincrease extracellular DA in the NACC. γ-Amino butyric acid (GABA)ergicneurons in the NACC and ventral pallidum project onto DA neurons in theVTA. Pharmacologic and electrophysiologic studies indicate theseprojections are inhibitory. Inhibition of VTA-DA neurons is likely theresult of GABA_(B) receptor stimulation. In addition, microinjection ofbaclofen into the VTA, acting via these receptor subtypes, can decreaseDA concentrations in the NACC. Taken together, it is evident thatpharmacologic manipulation of GABA may effect DA levels in the NACCthrough modulation of VTA-DA neurons.

Gamma vinyl GABA

Gamma vinyl GABA (GVG) is a selective and irreversible inhibitor ofGABA-transaminase (GABA-T) known to potentiate GABAergic inhibition. Itis also known that GVG alters cocaine's biochemical effects by causing adose-dependent and prolonged elevation of extracellular endogenous brainGABA levels.

GVG is C₆H₁₁NO₂ or 4-amino-5-hexanoic acid available as Vigabatrin® fromHoechst Marion Roussel and can be obtained from Marion Merell Dow ofCincinnati, Ohio. GVG does not bind to any receptor or reuptake complex,but increases endogenous intracellular GABA levels by selectively andirreversibly inhibiting GABA-transaminase (GABA-T), the enzyme thatnormally catabolizes GABA.

As used herein GVG includes the racemic compound or mixture whichcontains equal amounts of S(+)-gamma-vinyl GABA, and R(−)-gamma vinylGABA. This racemic compound of GVG is available as Vigabatrin® fromHoechst Marion Roussel and can be obtained from Marion Merell Dow ofCincinnati, Ohio.

GVG contains asymmetric carbon atoms and thus is capable of existing asenantiomers. The present invention embraces any enantiomeric form of GVGincluding the racemates or racemic mixture of GVG. In some cases theremay be advantages, i.e. greater efficacy, to using a particularenantiomer when compared to the other enantiomer or the racemate orracemic mixture in the methods of the instant invention and suchadvantages can be readily determined by those skilled in the art.

For example, the enantiomer S(+)-gamma-vinyl GABA is more effective atincreasing endogenous intracellular GABA levels than the enantiomerR(−)-gamma-vinyl GABA.

Different enantiomers may be synthesized from chiral starting materials,or the racemates may be resolved by conventional procedures which arewell known in the art of chemistry such as chiral chromatography,fractional crystallization of diastereomeric salts, and the like.

Administration of Gamma vinyl GABA

In living mammals (in vivo), GVG or pharmaceutically acceptable saltsthereof, can be administered systemically by the parenteral and enteralroutes which also includes controlled release delivery systems. Forexample, GVG can easily be administered intravenously, orintraperitoneal (i.p.) which is a preferred route of delivery.Intravenous or intraperitoneal administration can be accomplished bymixing GVG in a suitable pharmaceutical carrier (vehicle) or excipientas understood by practitioners in the art.

Oral or enteral use is also contemplated, and formulations such astablets, capsules, pills, troches, elixirs, suspensions, syrups, wafers,chewing gum and the like can be employed to provide GVG orpharmaceutically acceptable salts thereof.

As used herein, pharmaceutically acceptable salts include thosesalt-forming acids and bases which do not substantially increase thetoxicity of the compound. Some examples of suitable salts include saltsof mineral acids such as hydrochloric, hydriodic, hydrobromic,phosphoric, metaphosphoric, nitric and sulfuric acids, as well as saltsof organic acids such as tartaric, acetic, citric, malic, benzoic,glycollic, gluconic, gulonic, succinic, arylsulfonic, e.g.p-toluenesulfonic acids, and the like.

An effective amount as used herein is that amount effective to achievethe specified result of changing addiction-related behavior of themammal. It is an amount which will diminish or relieve one or moresymptoms or conditions resulting from cessation or withdrawal of thepsychostimulant, narcotic analgesic, alcohol, nicotine or combinationsthereof. It should be emphasized, however, that the invention is notlimited to any particular dose.

Preferably, GVG is administered in an amount which has little or noadverse effects. For example, the amount administered can be from about15 mg/kg to about 2 g/kg or from about 15 mg/kg to about 600 mg/kg.

For example, to treat cocaine addiction, GVG is administered in anamount of from about 15 mg/kg to about 2 g/kg, preferably from about 100mg/kg to about 300 mg/kg or from about 15 mg/kg to about 600 mg/kg andmost preferably from about 150 mg/kg to about 300 mg/kg or from about 75mg/kg to about 150 mg/kg.

To treat nicotine addiction, for example, GVG is administered in anamount of from about 15 mg/kg to about 2 g/kg or from about 15 mg/kg toabout 600 mg/kg, preferably from about 100 mg/kg to about 300 mg/kg orfrom about 150 mg/kg to about 300 mg/kg and most preferably from about18 mg/kg to about 20 mg/kg or from about 75 mg/kg to about 150 mg/kg.

To treat methamphetamine addiction, for example, GVG is administered inan amount of from about 15 mg/kg to about 2 g/kg, preferably from about100 mg/kg to about 300 mg/kg or from about l5 mg/kg to about 600 mg/kgand most preferably from about 150 mg/kg to about 300 mg/kg or fromabout 75 mg/kg to about 150 mg/kg to a mammal.

When the mammal is addicted to a combination of abused drugs, such asfor example, cocaine and heroin, GVG is administered in an amount offrom about 15 mg/kg to about 2 g/kg, preferably from about 100 mg/kg toabout 300 mg/kg or from about 15 mg/kg to about 600 mg/kg and mostpreferably from about 150 mg/kg to about 300 mg/kg or from about 75mg/kg to about 150 mg/kg to a mammal.

Mammals include, for example, humans, baboons and other primates, aswell as pet animals such as dogs and cats, laboratory animals such asrats and mice, and farm animals such as horses, sheep, and cows.

Gamma vinyl GABA (GVG) is a selective and irreversible inhibitor ofGABA-transaminase (GABA-T) known to potentiate GABAergic inhibition. Itis also known that GVG alters cocaine's biochemical effects by causing adose-dependent and prolonged elevation of extracellular endogenous brainGABA levels.

Based on the knowledge that cocaine, as well as other drugs of abuse,increases extracellular NACC DA and the fact that GABA inhibits DA inthe same nuclei, we have shown that GVG can attenuate cocaine, nicotine,methamphetamine, and ethanol-induced changes in extracellular DA. In oneexample, in vivo microdialysis techniques were used in freely movinganimals to show, the effects of acute (single injection) and chronic (11days) GVG administration on cocaine-induced increases in extracellularDA concentration in the NACC. See specifically Morgan, A. E., et al.“Effects of Pharmacologic Increases in Brain ABA Levels onCocaine—Induced Changes in Extracellular Dopamine,” Synapse 28:60-65(1998) the contents of which are incorporated herein as if set forth infull.

It has unexpectedly been found that intake of GVG alters behavior, andespecially addiction-related behavior associated with the biochemicalchanges resulting from intake of drugs of abuse. For example, GVGsignificantly attenuated cocaine-induced increases in neostriatalsynaptic DA in the primate (baboon) brain as assessed by positronemission tomography (PET) and abolished both the expression andacquisition of cocaine-induced conditioned place preference or CPP. Ithad no effect, however, on CPP for a food reward or on the delivery ofcocaine to the brain locomotor activity. These findings suggest thepossible therapeutic utility in cocaine addiction of a pharmacologicstrategy targeted at the GABAergic neurotransmitter system, a systemdistinct from but functionally linked to the DA mesotelencephalicreward/reinforcement system. However, rather than targeting the GABAreceptor complex with a direct GABA agonist, this novel approach withGVG takes advantage of the prolonged effects of an irreversible enzymeinhibitor that raises endogenous GABA levels without the addictiveliability associated with GABA agonists acting directly at the receptoritself.

Although GVG is used in the present examples, it will be understood bythose skilled in the arts that other compositions can be used which areknown to potentiate the GABAergic system or increase extracellularendogenous GABA levels in the CNS.

Such compositions include drugs which enhance the production or releaseof GABA in the CNS. These drugs include, but are not limited to,gabapentin, valproic acid, progabide, gamma-hydroxybutyric acid,fengabine, cetylGABA, topiramate, tiagabine, acamprosate(homo-calcium-acetyltaurine) or a pharmaceutically acceptable saltthereof, or an enantiomer or a racemic mixture thereof.

The present invention embraces any enantiomeric form of gabapentin,valproic acid, progabide, gamma-hydroxybutyric acid, fengabine,cetylGABA, topiramate, tiagabine, or acamprosate, including theracemates or racemic mixtures thereof.

As previously stated, in some cases there may be advantages, i.e.greater efficacy, to using a particular enantiomer when compared to theother enantiomer or the racemate or racemic mixture in the methods ofthe instant invention and such advantages can be readily determined bythose skilled in the art.

The present invention embraces compositions which include prodrugs ofGABA or drugs which contain GABA as a moiety in its chemical structure.These prodrugs become pharmacologically active when metabolically,enzymatically or non-enzymatically biotransformed or cleaved into GABAin the CNS. An example of a prodrug of GABA is progabide which, uponcrossing the blood brain barrier, increases endogenous CNS GABA levels.

As previously stated, Gamma vinyl GABA (GVG) is a selective andirreversible inhibitor of GABA-transaminase (GABA-T) known to potentiateGABAergic inhibition. Other compositions which inhibit GABA re-uptake inthe CNS are also encompassed by the present invention. An example of aGABA re-uptake inhibitor is tiagabine.

The method of the present invention is useful in potentiating theGABAergic system or increasing extracellular endogenous GABA levels inthe CNS. As used herein, enhancing or increasing endogenous CNS GABAlevels is defined as increasing or up-regulating GABA levelssubstantially over normal levels in vivo, within a mammal. Preferably,endogenous CNS GABA levels are enhanced at least by from about 10% toabout 600% over normal levels.

As previously stated, an effective amount as used herein is that amounteffective to achieve the specified result of changing addiction-relatedbehavior of the mammal. It is an amount which will diminish or relieveone or more symptoms or conditions resulting from cessation orwithdrawal of the psychostimulant, narcotic analgesic, alcohol, nicotineor combinations thereof. It should be emphasized, however, that theinvention is not limited to any particular dose.

For example, an effective amount of gabapentin administered to themammal is an amount from about 500 mg to about 2 g/day. Gabapentin isavailable as Neurontin® from Parke-Davis in the United States.

An effective amount of valproic acid administered to the mammal, forexample, is preferably, an amount from about 5 mg/kg to about 100mg/kg/day. Valproic acid is available as Depakene® from Abbott in theUnited States.

Preferably, an effective amount of topiramate administered to the mammalis, for example, an amount from about 50 mg to about 1 g/day. Topiramateis available as Topamax® from McNeil in the United States. An effectiveamount of progabide administered to the mammal is, preferably, an amountfrom about 250 mg to about 2 g/day. Progabide is available as Gabrene®from Synthelabo, France. The chemical formula of progabide isC₁₇H₁₆N₂O₂.

An effective amount of fengabine administered to the mammal is,preferably, an amount from about 250 mg to about 4 g/day. Fengabine isavailable as SL 79229 from Synthelabo, France. The chemical formula offengabine is C₁₇H₁₇C₁₂NO.

Preferably, an effective amount of gamma-hydroxybutyric acidadministered to the mammal is an amount from about 5 mg/kg to about 100mg/kg/day. Gamma-hydroxybutyric acid is available from Sigma Chemical.The chemical formula of gamma-hydroxybutyric acid is C₄H₇O₃Na.

Details of the invention have been set forth herein in the form ofexamples which are described below. The full scope of the invention willbe pointed out in the appended claims.

EXAMPLES

Examples have been set forth below for the purpose of illustration andto describe the best mode of the present invention. The scope of theinvention is not to be in any way limited by the examples set forthherein.

Materials and Methods 1. Primate PET Studies

Twenty adult female baboons (Papio anubis, 13-18 kg) were used for allstudies and carbon-11 labeled raclopride, previously shown to besensitive to changes in synaptic DA was synthesized as previouslydescribed (Volkow, et al., 1994). Arterial blood samples were obtainedthroughout the study and selected plasma samples were analyzed for thepresence of unchanged radio tracer carbon-11. Animals were not removedfrom the gantry between isotope injections. Regions of interest (ROI's)were drawn directly on the PET images. Briefly, the corpus striatum wasoutlined, bilaterally, on every transaxial slice upon which it appeared.The cerebellar ROI was drawn across the midline at the level of thecerebellar vermis. ROI's from the first study were then copied directlyonto the corresponding slice from the second. By examining placement ofthe ROI's on the second scan changes could be made, if necessary, in ROIposition only. This multi planar method of analysis reduced differencesthat may arise due to movement of the animal within the gantry duringthe scanning interval.

A graphical method for determining the distribution volume (DV) wasdeveloped previously for the kinetic analysis of the [¹¹C]-raclopridedata. The DV ratio was the most reproducible measure of racloprideuptake. The ratio is the DV from a receptor-rich region (corpusstriatum) to the DV of a non-receptor region (cerebellum). The freereceptor concentration was directly proportional to the DV ratio of 1.Animal preparation was conducted as detailed previously (Dewey, et al.,1992).

The statistical analysis was designed to test the hypothesis that (1)the cocaine challenge differed from the test/retest variability of theradio tracer carbon-11 (performed in the same animals under identicalexperimental conditions) and (2) the challenge conditions differed fromeach other. The fact that significant results were obtained for thestriatum and striatum to cerebellum ratio, but not for the cerebellum,indicated that the effects of the intervention were limited to thespecific, but not the non-specific binding component. GVG did not alterthe regional distribution nor the rate of metabolism of the radiotracer.

2. Cocaine-Induced Conditioned Place Preference in Rodents

In all rodent studies, male Sprague-Dawley rats were used (200-225 g,Taconic farms, Germantown, N.Y.). Animals were allowed to acclimate tothe animal housing facility for at least 5 days prior to beginning theexperiments. We used conditioned place preference (CPP) chambers aspreviously described (Lepore et al., 1995), except instead of onechamber being entirely white and the other black, one chamber wasentirely light blue with a stainless steel floor and the second chamberwas light blue with horizontal black stripes (2.5 cm wide) spaced 3.8 cmapart with a smooth plexiglass floor. In all CPP studies with GVG, thesaline volume was (1 ml/kg), and the cocaine doses were 20 mg/kg. Thesaline, cocaine and GVG were all injected intraperitonealy (i.p.). Theconditioning procedure for the acquisition phase consisted of 12sessions carried out consecutively over 12 days.

The CPP pairings were: 1) saline/saline 2) saline/cocaine 3) GVG/saline4) saline/cocaine and GVG. The animals in each group were randomlyassigned to a 2×2 factorial design with one factor being the pairingchamber and the other factor being the order of conditioning. Theanimals that received either saline or cocaine were injected andconfined to the appropriate compartment for 30 minutes. The GVGinjections were given 3 hours before saline or cocaine injection andsubsequent placement of the animals in the appropriate chamber. This wasdone as it has been shown that GABA levels reach maximal values 3 to 4hours following GVG administration.

On the test day (day 12), neither drugs nor saline were administered andthe animal was allowed to move freely between both chambers for fifteenminutes. The amount of time spent in each chamber was recorded using anautomated infrared beam electronically coupled to a timer. For theexpression phase of CPP to cocaine, the animals were habituated andconditioned to cocaine as described in the acquisition studies, but noanimals in the expression studies were given GVG on conditioning days.On the test day (day 12), the animals being tested in the expressionphase, unlike the animals in the acquisition phase, received eithersaline or GVG 2.5 hours before they were placed in the apparatus andallowed free access to both chambers for 15 minutes.

3. Food-Induced Conditioned Place Preference in Rodents

In order to test food-induced CPP in rodents, four groups of rats wereallowed access to food ad libitum during the entire 12 session of CPPprocedure. The 12 session CPP procedure was exactly the same as theprocedure used in the cocaine induced CPP studies except the appetitivesubstance was food rather than cocaine. Group one was given saline,group two was given intraperitoneally 150 mg/kg of GVG, group 3 wasgiven saline and group 4 was given intraperitoneally 300 mg/kg of GVGprior to food exposure and CPP pairing to a side of the CPP box. Theanimals in all four groups were habituated to Froot Loops, afruit-flavored breakfast cereal that is very appealing to laboratoryrats, in the appropriate chamber in the test room during fourhabituating sessions. Twenty-four hours after the last CPP pairing, theanimals were placed in the chamber and neither drug nor saline (norfood) was administered (nor available) and animals were allowed to movefreely within the CPP apparatus for 15 minutes. The amount of time spentin the paired and unpaired chambers was recorded using an automateddevice.

4. Locomotor Activity Measured in Rodents

Animals were prehandled for 5 minutes each day for one week prior to theexperiment to reduce handling stress. On the day of the study, GVG (150mg/kg or 300 mg/kg) or saline (1 ml/kg or 0.5 ml/kg) was administeredintraperitoneally 2.5 hours prior to the experiment. The animals weretransported to the testing area one hour before each experiment. 2.5hours after GVG or saline administration, animals were placed in thebehavior cages and the locomotor activity was recorded in 10 minuteintervals for 90 minutes onto a PC-AT computer using the hardware forthe Photobeam Activity System. The locomotor cages themselves are41.3×41.3×30.5 cm clear acrylic cages. The electronic system (PhotobeamActivity system, San Diego Instruments, San Diego, Calif.) used tomonitor locomotor activity consists of 16 infrared beams projectingacross the cages from left to right and 16 beams from front to back. Allthe infrared beams are approximately 0.39 cm from the floor.

5. Catalepsy Studies in Rodents

The degree of catalepsy following the administration intraperitoneallyof 150 mg/kg GVG, 300 mg/kg intraperitoneally GVG or saline (1 ml/kg,i.p. 0.9% saline) was determined by using the Bar test. Briefly, maleSprague-Dawley rats were handled and transported to the test room threedays prior to the experiments to allow for acclimation. On the test day,the animals (n=10 per treatment group) received either saline or GVG,and the degree of catalepsy was measured 60, 120 and 240 minutesfollowing injection. The experimenter was blind to the treatmentreceived by each animal. The bar was composed of wood and had a diameterof 1.2cm and height from floor to the top of the bar was 10cm. For eachdetermination, the forepaws of the animals were gently placed over thebar and the time it took the animal to move both forepaws to the floorwas measured.

6. [¹¹C]-Cocaine Studies in Rodents and Primates

Animals (n=10) were placed into two groups. In group 1, saline (1 ml/kg)was administered via intraperitoneal (i.p.) injection 3 hours prior toi.p. [¹¹C]-cocaine administration. In group 2, GVG (300 mg/kg) wasadministered via i.p. injection 3 hours prior to i.p. [¹¹C]-cocaineadministration. Animals were sacrificed 10 minutes following[¹¹C]-cocaine injection. Brains were removed and counted forradioactivity. In two additional primate PET studies, GVG wasadministered (300 mg/kg) immediately following a baseline scan withlabeled cocaine. Approximately 3 hours later, labeled cocaine was againadministered and animals were scanned for 60 minutes.

7. Microdialysis studies in Rodents

All animals were used under an IACUC-approved protocol and with strictadherence to the NIH guidelines. Adult male Sprague-Dawley rats (200-300g, Taconic Farms), housed in the animals care facility under 12:12light/dark conditions, were placed into 6 groups (n=5-9), anesthetizedand siliconized guide cannulae were stereotactically implanted into theright NACC (2.0 mm anterior and 1.0 mm lateral to bregms, and 7.0 mmventral to the cortical surface) at least 4 days prior to study.Microdialysis probes (2.0 mm, Bioanalytical Systems, BAS, WestLafayette, Ind.) were positioned within the guide cannulae andartificial cerebrospinal fluid (ACSF, 155.0 mM NA−, 1.1 mM Ca²⁻, 2.9 mMK⁻, 132.76 mM Cl⁻, and 0.83 mM Mg²⁻) was administered through the probeusing a CMA/100 microinfusion pump (BAS) at a flow rate of 2.0 μl/min.Animals were placed in bowls, and probes were inserted and flushed withACSF overnight. On the day of the study, a minimum of three samples wereinjected to determine baseline stability. Samples were collected for 20min. and injected on-line (CMA/160, BAS). The average Dopamineconcentration of these three stable samples was defined as control(100%), and all subsequent treatment values were transformed to apercentage of that control. Upon establishing a stable baseline, thenicotine was administered by intraperitoneal (i.p.) injection. The highperformance liquid chromatography (HPLC) system consists of a BASreverse-phase column (3.0 μC-18), a BAS LC-4C electrochemical transducerwith a dual/glassy carbon electrode set at 650 mV, a computer thatanalyzes data on-line using a commercial software package (ChromographBioanalytical Systems), and a dual pen chart recorder. The mobile phase(flow rate 1.0 ml/min) consisted of 7.0% methanol, 50 mM sodiumphosphate monobasic, 1.0 mM sodium octyl sulfate, and 0.1 mm EDNA, pH4.0. DA eluted at 7.5 min. Upon completion of the study, animals weredecapitated and frozen sections were obtained for probe placementverification.

In parallel to the quantitative estimates of dopamine concentration, thelocomotor response of these animals to stimulant administration wassimultaneously quantified using an infrared motion sensor. This infraredoptical proximity detector monitored movement of the gimbaled arm, anintegral component of the freely moving system. The digital output ofthe detector was interfaced with an IBM personal computer and programmedto count both positive and negative arm deflections. These data werecollected and totaled using the same temporal sampling protocol used forthe dialysis samples. Locomotor activity was then expressed as thenumber of deflections per sample interval.

Example 1 Non-Human Primate (Baboon) Studies

In this example twenty non-human primates received two [¹¹C]-racloprideinjections in accordance with the procedure described in Section 1 ofMaterials and Methods. The first served as a baseline and the secondfollowed cocaine or placebo. Test/retest primates (n=7) shown as Group 1of Table 1 below received placebo (0.9% saline, 1 ml/kg) prior to thesecond radio tracer injection in order to determine the test/retestvariability of this imaging method.

TABLE I Groups and experimental conditions Group Pharmacologic condition1 Control (test/retest) 2 Cocaine treated 3 GVG/Cocaine treated

All remaining primates (n=13) received a systemic injection of cocainehydrochloride (0.5, 1.0 or 2.0 mg/kg) either 5 or 30 minutes prior tothe second [¹¹C]-raclopride injection. Of these 13 animals, fivereceived GVG (300 mg/kg, iv) 3 hours prior to cocaine administration.

Different cocaine doses and cocaine pretreatment time intervals producedno significant changes in the effects of cocaine on the distributionvolume (DV), in line with expectations. Thus, the average % change inthe DV ratio for animals treated with cocaine alone (n=8) versusGVG/cocaine (n=5) as Groups 2 and 3 of FIG. 1 respectively.

As a competitive antagonist, [¹¹C]-raclopride's binding is dependentupon the concentration of DA in the synaptic cleft. That is, as synapticDA concentrations decrease, [¹¹C]-raclopride binding increases.Conversely, as synaptic DA concentrations increase, [¹¹C]-raclopridebinding decreases. As seen in FIG. 1, the test/retest variability ofthis imaging method was less than 7% for group 1. The variability ofthese PET measurements is consistent with previous values obtained with[¹¹C]-raclopride in primates. In Group 2, cocaine produced a greaterthan 30% reduction in the mean DV ratio (p<0.0002, Student's two-tailedt-test, FIG. 1). These data are consistent with simultaneous PET andmicrodialysis studies in which an amphetamine challenge increasedextracellular DA and decreased [¹¹C]-raclopride binding in the primatebrain. In addition, these findings are similar to a recent report whichexamined the effects of a cocaine challenge on labeled raclopridebinding in the human. Finally, these data are consistent with our ownmicrodialysis studies (Morgan and Dewey, 1998) as well as our primateand human PET studies with amphetamine, GBR 12909, tetrabenazine,methylphenidate, and [¹¹C]-raclopride (Dewey et al., 1993; Volkow, etal., 1994). GVG pretreatment, however, significantly blocked thecocaine-induced decrease as shown in Group 2 of FIG. 1 in the DV ratio(group 2, p<0.002, Student's two-tailed t-test). These differences arereadily apparent in the parametric DV ratio images as shown in FIG. 2.Values for groups 1 and 3 were not statistically different (p>0.1,Student's two-tailed t-test).

Example 2 Cocaine-Induced Conditioned Place Preference Studies inRodents

In this example the procedure outlined in Section 2 of Materials andMethods was followed. Cocaine produced a dose-dependent CPP response,with the most reliable and robust response occurring at 20 mg/kg asshown in Table 2 below.

TABLE II Conditioned place preference to cocaine Time spent in chambers(mins) Cocaine (mg/kg) Paired Unpaired¹ 0 7.4 ± 0.3  7.6 ± 0.3 5.0 8.2 ±0.4  6.8 ± 0.5 10.0 9.6 ± 0.5² 5.4 ± 0.3 20.0 11.8 ± 0.4³   3.2 ± 0.4⁴¹Monitored animals were injected only with saline ²Significantly greaterthan the 0 and 5 mg/kg doses of cocaine, p < 0.05, analysis of variance(ANOVA) and Student-Newman-Keuls test. ³Significantly greater than the0.5 and 10 mg/kg doses of cocaine, p < 0.05, ANOVA andStudent-Newman-Keuls test. ⁴Significantly less than 0.5 and 10 mg/kgdoses of cocaine, p < 0.01, ANOVA and Student-Newman-Keuls test.

We therefore chose a 20 mg/kg cocaine dose with which to examine theeffect of GVG administration on the acquisition and expression phases ofcocaine-induced CPP. The results clearly indicated that 112, 150 and 300mg/kg, but not 75 mg/kg, of GVG blocked the acquisition and expressionof cocaine-induced CPP. See specifically Tables 3-10 below.

TABLE III Effect of GVG and saline on the acquisition of cocaine inducedconditioned place preference Time spent in chambers (min) Treatmentpairings¹ Paired Unpaired² Saline/Saline 7.3 ± 0.5 7.7 ± 0.6Saline/Cocaine 11.1 ± 0.3⁴ 3.9 ± 0.4 75 mg/kg GVG³/Saline 7.3 ± 0.5 7.7± 0.6 75 mg/kg GVG³/Cocaine 9.1 ± 1.1 5.9 ± 1.2 ¹Each value representsthe mean number of minutes spent in each chamber ± S.E.M. (n − 8-10).²Monitored animals were injected only with saline. ³Animals received GVGor Saline 2.5 hours prior to receiving saline or cocaine (20 mg/kg).⁴Significantly greater than all treatment groups , p < 0.05, ANOVA andNewman-Keuls Test. ⁵Significantly less than all treatment groups, p <0.01, ANOVA and Newman-Keuls test.

TABLE IV Time spent in chambers (mins) Treatment pairings¹ PairedUnpaired² Saline/Saline 7.2 ± 0.5 7.8 ± 0.4 Saline/Cocaine 11.8 ± 0.5⁴3.2 ± 0.5 112 mg/kg GVG³/Saline 7.6 ± 0.6 7.4 ± 0.6 112 mg/kgGVG³/Cocaine 8.2 ± 0.5 6.8 ± 0.5 ¹Each value represents the mean numberof minutes spent in each chamber ± S.E.M. (n = 8-10). ²Monitored animalswere injected only with saline. ³Animals received GVG or Saline 2.5hours prior to receiving saline or cocaine (20 mg/kg). ⁴Significantlygreater than all treatment groups, p < 0.05, ANOVA and Newman-KeulsTest. ⁵Significantly less than all treatment groups, p < 0.01, ANOVA andNewman-Keuls test.

TABLE V Time spent in chambers (min) Treatment pairings¹ PairedUnpaired² Saline/Saline 7.4 ± 0.3 7.6 ± 0.4 Saline/Cocaine 11.6 ± 0.5⁴ 3.4 ± 0.4⁵ 150 mg/kg GVG³/Saline 7.8 ± 0.6 7.2 ± 0.6 150 mg/kgGVG³/Cocaine 7.9 ± 0.8 7.1 ± 0.8 ¹Each value represents the mean numberof minutes spent in each chamber = S.E.M. (n = 8-10). ²Monitored animalswere injected only with saline. ³Animals received GVG or Saline 2.5hours prior to receiving saline or cocaine (20 mg/kg). ⁴Significantlygreater than all treatment groups, p < 0.05, ANOVA and Newman-KeulsTest. ⁵Significantly less than all treatment groups, p < 0.01, ANOVA andNewman-Keuls Test.

TABLE VI Time spent in chambers (mins) Treatment pairings¹ PairedUnpaired² Saline/Saline 7.7 ± 0.3 7.3 ± 0.3 Saline/Cocaine 11.2 ± 0.6⁴ 3.8 ± 0.5⁵ 300 mg/kg GVG³/Saline 7.2 ± 0.4 7.8 ± 0.4 300 mg/kgGVG³/Cocaine 7.6 ± 0.7 7.2 ± 0.7 ¹Each value represents the mean numberof minutes spent in each chamber ± S.E.M. (n = 8-10). ²Monitored animalswere injected only with saline. ³Animals received GVG or Saline 2.5hours prior to receiving saline or cocaine (20 mg/kg). ⁴Significantlygreater than all treatment groups, p < 0.05, ANOVA and Newman-KeulsTest. ⁵Significantly less than all treatment groups, p < 0.01, ANOVA andNewman-Keuls Test.

TABLE VII Effect of GVG and saline on the expression of cocaine-inducedconditioned place preference Treatment Drug given on Time spent inchambers (min) pairings¹ test day Paired Unpaired² Saline/Saline Saline 7.5 ± 0.4¹ 7.5 ± 0.4 Saline/Saline GVG, 75 mg/kg 7.5 ± 0.3 7.5 ± 0.3Saline/Cocaine Saline 11.8 ± 0.5³ 3.2 ± 0.5 Saline/Cocaine GVG, 75 mg/kg10.6 ± 0.6³ 4.4 ± 0.9 Saline/Saline Saline  7.8 ± 0.5¹ 7.2 ± 0.6 ¹Eachvalue represent the mean number of minutes spent in each chamber ±S.E.M. (n = 10). ²Monitored animals were injected only with saline.³Significantly greater than all other treatment paintings, p < 0.01,ANOVA and Student Newman-Keuls test.

TABLE VIII Treatment Drug given on Time spent in chambers (min)pairings¹ test day Paired Unpaired² Saline/Saline Saline 7.1 ± 0.5 7.9 ±0.5 Saline/Saline GVG, 112 mg/kg 7.2 ± 0.3 7.8 ± 0.3 Saline/CocaineSaline 12.2 ± 0.6³ 2.8 ± 0.5 Saline/Cocaine GVG, 112 mg/kg 8.1 ± 0.7 6.9± 0.6 ¹Each value represents the mean number of minutes spent in eachchamber ± S.E.M. (n = 10). ²Monitored animals were injected only withsaline. ³Significantly greater than all other treatment pairings, p <0.01, ANOVA and Student Newman-Keuls test.

TABLE IX Treatment Drug given on Time spent in chambers (min) pairings¹test day Paired Unpaired² Saline/Saline Saline  7.2 ± 0.2¹ 7.8 ± 0.2Saline/Saline GVG, 150 mg/kg 7.7 ± 0.2 7.3 ± 1.1 Saline/Cocaine Saline11.1 ± 0.5³  3.9 ± 0.4⁴ Saline/Cocaine GVG, 150 mg/kg 7.9 ± 0.3 7.1 ±0.3 ¹Each value represents the mean number of minutes spent in eachchamber ± S.E.M. (n = 10). ²Monitored animals were injected only withsaline. ³Significantly greater than all other treatment pairings, p <0.01, ANOVA and Student Newman-Keuls test. ⁴Significantly less than allother treatment pairing, p < 0.01, ANOVA and Student Newman-Keuls test.

TABLE X Treatment Drug given on Time spent in chambers (min) pairings¹test day Paired Unpaired² Saline/Saline Saline  7.8 ± 0.5¹ 7.2 ± 0.6Saline/Saline GVG, 300 mg/kg 7.3 ± 0.4 7.7 ± 0.3 Saline/Cocaine Saline12.5 ± 0.8³  2.5 ± 0.6⁴ Saline/Cocaine GVG, 300 mg/kg 7.9 ± 0.5 7.1 ±0.6 ¹Each value represents the mean number of minutes spent in eachchamber ± S.E.M. (n = 10). ²Monitored animals were injected only withsaline. ³Significantly greater than all other treatment pairings, p <0.05, ANOVA and Student Newman-Keuls test. ⁴Significantly less than allother treatment pairings, p < 0.05, ANOVA and Student Newman-Keuls test.

By itself, GVG produced neither a CPP nor a conditioned aversiveresponse. See again, Tables 3-10.

Example 3 Food-Induced Conditioned Place Preference Studies in Rodents

In this example the procedure outlined in Section 3 of Materials andMethods was followed. The results set forth in Table 11 below indicatethat food elicited an incentive or rewarding effect. For example, allpaired values show that rodents spent more time in the chamber wherefood was present.

TABLE XI Effect of GVG (150, 300 mg/kg,ip) on conditioned placepreference to food Time spent in chambers (min) Treatment pairingsPaired Unpaired² Saline/Saline 7.3 ± 0.6 7.7 ± 0.6 GVG/Saline 7.5 ± 0.77.5 ± 0.7 Saline/Food 9.3 ± 0.7 5.7 ± 0.7 GVG (150 mg/kg)/Food 9.4 ± 0.45.6 ± 0.5 GVG (300 mg/kg)/Food 9.0 ± 0.5 6.0 ± 0.5 ¹Each valuerepresents the mean number of minutes spent in each chamber ± S.E.M²Monitored animals were injected only with saline.

The administration of 150 or 300 mg/kg of GVG did not alter the CPPresponse to food as shown in Table 11 despite attenuating the incentivemotivational effects of cocaine in the above noted CPP experiments asshown in Tables 3-10 above.

Discussion of Experimental Results Obtained in Examples 1, 2 and 3

In previous PET studies, we showed that GVG alone reduces extracellularDA concentrations resulting in an increase in [¹¹C]-raclopride bindingin the primate brain (Dewey, et al., 1992). In the PET studies of thepresent invention, GVG-induced decreases in extracellular DA levelsprior to cocaine administration clearly underlie the attenuation ofcocaine's effects observed in group 3 of Table 1. However, the seeminglyidentical values found for groups 1 and 3, combined with our previousfindings using GVG alone (Dewey, et al., 1992), indicate that cocaineincreased extracellular DA levels in the present invention despite GVGadministration, but only to baseline values.

However, based on the CPP data presented here, this cocaine-inducedreturn to baseline was apparently insufficient to produce incentivemotivational effects. Our results indicate that cocaine produced a CPPresponse. In contrast, vehicle pairings did not produce a CPP response,indicating that the animals did not display a chamber preference, i.e.,the apparatus is unbiased. In addition, the CPP response to cocaine wasdose-dependent, with the most reliable and robust response occurring atthe 20 mg/kg cocaine dose.

Administration of 112, 150, 300 mg/kg but not 75 mg/kg of GVG blockedthe acquisition and expression of the CPP response elicited by cocaine.In contrast, GVG, when paired with saline, did not produce a CPP oraversive response. This indicates that the blockade of the CPP tococaine by GVG was not related to GVG's eliciting an aversive orappetitive response by itself. Our results presented in Example 2indicated that food elicits an incentive or rewarding effect. Theadministration of 150 or 300 mg/kg of GVG did not alter the CPP responseto food, despite attenuating the incentive effects of cocaine. Thisfinding suggests that GVG specifically attenuates therewarding/incentive effects of cocaine.

Example 4 Locomotor Activity and Catalepsy Studies in Laboratory Rodents

In this example the procedures outlined in Section 4 and 5 of Materialsand Methods were followed. Although it is widely accepted that the CPPparadigm differentiates incentive motivational effects from motoriceffects, we nevertheless assessed GVG's effects on locomotion andcatalepsy in rats. We found that pretreatment with GVG at doses of 150mg/kg or 300 mg/kg did not alter locomotor activity compared to salinepretreated controls as shown in FIGS. 3a and 3b. In addition,pretreatment with GVG at doses of 150 mg/kg or 300 mg/kg did not inducecatalepsy in rats. Catalepsy duration after 300 mg/kg GVG was 1.1+0.4seconds (n=10), which was not significantly different from 0.7+0.3seconds (n=10) in saline-treated rats. n indicates the number of rodentswhich were tested.

Example 5 ¹¹C-Cocaine levels in Rodents and Primates

In this example the procedure outlined in Section 6 of Materials andMethods was followed. In order to assess the possibility that GVG couldattenuate cocaine's actions by altering its penetration into the brain,we examined the effect of saline and GVG on [¹¹C]-cocaine levels in thewhole rat and primate brain. In rodents, the levels of [¹¹C]-cocaine inthe brain following intraperitoneal administration of saline and 300mg/kg GVG were 0.110±0.03 and 0.091±0.02, respectively, which did notstatistically differ. In primates, the pharmacokinetic profile oflabeled cocaine binding in the neostriatum was not significantlydifferent from the baseline scan both in terms of absolute uptake aswell as clearance.

Example 6

In this example, the effects of GVG on nicotine-induced changes inextracellular dopamine concentrations were measured in freely movingrats. The procedure outlined in Section 7 of Materials and Methods wasfollowed.

A total of 8 rats were examined for each treatment pairing. Animalsreceived 4 pairings over an 8 day period, one pairing per day. Animalsreceived 75 mg/kg of GVG 2.5 hours prior to receiving 0.4 mg/kg ofnicotine. Animals were given GVG, then nicotine and placed in theappropriate chamber on day 1. On day 2, the animals were given GVG, thensaline and placed in the appropriate chamber. The protocol on days 1 and2 was repeated 3 additional times. Twenty four hours after the lastpairing was administered, the animals were allowed free access to theentire behavioral apparatus for 15 minutes and the amount of time spentin the paired and unpaired chambers recorded using an automated device.The effects of 75 mg/kg of intraperitoneally applied GVG on acquisitionof CPP to nicotine by the rats examined in this example is set forth inTable XII below.

TABLE XII Effect of 75 mg/kg i.p. GVG on acquisition of conditionedplace preference to (−)-nicotine. Time spent in chambers (min)¹Treatment Pairings Paired Unpaired² Nicotine 0.4 mg/kg, 9.4 ± 0.5  5.6 ±0.5  s.c./Vehicle³ 75 ,g/kg GVG/Nicotine, 6.4 ± 0.3⁴ 8.6 ± 0.3⁵ 0.4mg/kg, s.c. ¹Each value represents the mean number of minutes spent ineach chamber ± S.E.M. ²Monitored animals were injected only with saline.³The vehicle was 1 ml/kg of 0.9% NaCl or saline solution. ⁴Significantlyless than nicotine/vehicle pairing, p < 0.01, ANOVA andStudent-Newman-Keuls test. ⁵Significantly greater than nicotine/vehiclepairing, p < 0.01; ANOVA and Student-Newman-Keuls test.

The results of a similar experiment as the one summarized in Table XIIare shown in FIG. 4. FIG. 4 shows that GVG (150 mg/kg) blocksnicotine-induced increases in dopamine concentrations in freely movingrats. The open circles are control animals. The closed circles are fromanimals treated with GVG 2.5 hours before nicotine.

Comparative Example Effects of Baclofen on Cocaine Use

Our results obtained in Examples 1, 2 and 3 were consistent withprevious studies suggesting that the augmentation of GABAergic functioncan attenuate the rewarding/reinforcing actions of cocaine and otherdrugs of abuse. For example, it has been shown that, using theprogressive ratio paradigm, the selective GABA_(B) agonist baclofenproduced a dose-dependent decrease in the break points for intravenous(i.v.) administration of cocaine in male Wistar rats, although it didnot affect the rate of drug intake. These results suggested thatbaclofen attenuated the reinforcing effects of cocaine, as a decrease inthe break point represents a decrease in the motivation toself-administer cocaine.

It has also been hypothesized that augmentation of GABA_(A) receptorfunction carry attenuate cocaine self-administration, aschlordiazepoxide and alprazolam, positive allosteric modulators of theGABA_(A) receptor complex, decreased the rate of cocaineself-administration. However, this effect is probably related to anincrease in the reinforcing value of each unit dose of cocaine, aschlordiazepoxide will increase the break point for cocaineself-administration on a progressive ratio schedule.

The findings with baclofen were reinforced by a recent study from thesame laboratory indicating that acute pretreatment of rats with baclofen(1.25-5 mg/kg i.p.) will suppress self-administration of cocaine in adiscrete trials paradigm for at least four hours without significantlyaltering responding for food reinforcement. Microinjection of baclofeninto the ventral tegmental area ipsilateral to a stimulating electrodein the lateral hypothalamus of rats produced a rightward shift of therate-current intensity curve, indicating that baclofen attenuated therewarding value of the electrical stimulation. However, baclofen did notaffect the maximal responding rate for electrical brain stimulationreward or non-reinforced performance levels, suggesting that baclofen'saction was not related to alterations in motor performance/dexterity.

A recent study demonstrated that GVG produced a dose-dependent increasein brain stimulation reward thresholds in male F344 rats (Kushner etal., 1997b), without significant effects on motor performance. Thedecrease in brain stimulation reward thresholds produced by 2.5 and 5mg/kg of intraperitoneally administered cocaine was significantlyantagonized by 400 mg/kg dose of GVG.

Finally, the CPP response elicited by morphine (8 mg/kg) wassignificantly attenuated by microinjection of baclofen (0.1-1 nmol) intothe ventral tegmental area and this effect was antagonized by theGABA_(B) antagonist 2-hydroxysaclofen. Thus, despite using differentparadigms to assess reward/reinforcement, these studies indicate thatactivation of GABA_(B) receptors attenuated the appetitive value ofcocaine, morphine and electrical brain stimulation reward.

Previously, it was reported that pretreatment with the GABA-mimeticcompound progabide (which augments GABA levels in the brain via itsmetabolism to GABA), which alone does not produce conditioned placepreference or aversion, did not alter the CPP response to 1.5 mg/kg i.p.of amphetamine. However, it is difficult to compare this finding to thepresent invention as there were differences in rat strains, GABAergiccompounds and drugs used to elicit CPP. It should also be noted thatprogabide was only present for 35 minutes. Since it has been shown thatthe maximal increase in GABA levels in the brain following systemicprogabide occurs four-six hours after injection, GABA levels were not attheir maximum during the determination of amphetamine-induced CPP.

Given the evidence suggesting that augmentation of dopaminergic functionin the mesolimbic system plays a role in mediating therewarding/reinforcing effects of cocaine, the abolition of the CPPresponse to cocaine by GVG may be related to an alteration ofdopaminergic activity/function. This hypothesis is supported by our invivo microdialysis study indicating that acute (300 and 500 mg/kg i.p.)or repeated administration (100, 300, and 500 mg/kg i.p.) of GVGproduced a significant dose-dependent decrease in the elevation ofextracellular DA levels in the NACC and striatum produced by 20 mg/kgi.p. of cocaine (Dewey, et al., 1998). At the same time, it is unlikelythat an alteration in the sensitivity of DA receptors following GVGadministration is responsible for its attenuation of cocaine's action,because it is known that the repeated administration of GVG does notalter D₁ or D₂ receptor sensitivity in the rat striatum. However, noevidence exists regarding GVG's effects on other DA receptors (D₃, D₄and D₅). Alternatively, it is possible that cocaine could alter GABA_(B)receptor function, thereby potentially altering the release ofneurotransmitters such as DA and this could be antagonized by GVG viaelevation of GABA levels and consequent stimulation of GABA_(B)receptors.

It has also been shown that the repeated administration of cocainediminishes the effectiveness of presynaptic GABA_(B) auto andhetero-receptors on lateral septal nucleus neurons in rat brain slices.This may lead to a disinhibtory action and enhanced neurotransmitterrelease. It is also possible that baclofen could attenuate the action ofDA and this would attenuate cocaine's actions. This is indirectlysupported by the findings of Lacey et al. (1988), showing that inintracellular recordings from rat substantia nigra zona compactaneurons, the outward currents elicited by DA were occluded by maximalcurrents produced by baclofen.

Several interpretations of the present results are possible. First, itis possible that GVG could increase the metabolism of cocaine, therebydecreasing the amount which reaches the brain and subsequentlydiminishing its neurochemical effects and ultimately its behavioralactions. However, this is unlikely as brain levels of ¹¹C-cocaine werenot significantly altered in rats or primates pretreated with GVG (300mg/kg). Furthermore, cocaine is primarily metabolized by plasmacholinesterases whereas GVG is excreted primarily unchanged in theurine, making a pharmacokinetic interaction unlikely.

It has been reported that drugs which augment GABAergic function canproduce sedation and ataxia. Consequently, it is reasonable to postulatethat GVG, by producing such adverse behavioral effects, maynon-specifically antagonize cocaine's action. However, the results inthe present study indicate that GVG does not produce catalepsy orsignificantly alter locomotor activity, making this hypothesisuntenable. Furthermore, the examples discussed above show that GVG doesnot produce conditioned place aversion, indicating that its antagonismof cocaine's action is not the result of a counterbalancing aversiveaction. In addition, GVG does not elicit CPP alone, indicating that itis not shifting the preference of animals from the cocaine-paired to theGVG-paired environment.

It has been shown that GVG administration can alter food consumption inrats. Based on this, it is possible that GVG may decrease or attenuatethe hedonic value of natural rewards, as well as that elicited bycocaine. However, the present study shows that neither 150 nor 300 mg/kgof GVG alters CPP to food.

There is evidence indicating that behavior in the conditioned placepreference (CPP) paradigm depends upon both the affective andmemory-improving properties of the reinforcers under test. Therefore,one might argue that GVG's blockade of the expression and acquisition ofcocaine-induced CPP is the result of GVG interfering with theassociation of cocaine-induced positive incentive value with theappropriate stimuli by interfering with memory. Indeed, it is known thatcertain drugs which augment GABAergic function can impair memory.However, GVG does not affect place conditioning for food, suggestingthat this hypothesis cannot explain GVG's antagonism of cocaine's actionin the CPP paradigm.

It has been found that the 112, 150 and 300 mg/kg doses of GVGantagonize the acquisition and expression of cocaine-induced CPP. Incontrast, GVG did not elicit a CPP or conditioned place aversionresponse, indicating that GVG does not antagonize cocaine's action byproducing a CPP response alone or by attenuating CPP by producing anaversive effect. Furthermore, GVG did not elicit catalepsy and did notalter the incentive value of food. There is evidence thatcocaine-related stimuli or cues will reinstate drug-seeking behavior andcraving in detoxified cocaine addicts, thereby leading to relapse. Theexpression of the CPP to cocaine, determined in the absence of cocaine,is antagonized by GVG. These results indicate that the cravingexperienced by cocaine addicts can be attenuated by GVG.

Dopaminergic transmission in the NACC has been specifically implicatedin the reinforcing properties of cocaine. In the PET studies discussedabove, measurements were made in the corpus striatum rather than theNACC. Although DA neurotransmission in the corpus striatum has not beenimplicated in cocaine reward and reinforcement, the effects of cocaineon extracellular DA levels are qualitatively similar in both areas. Inaddition, our in vivo microdialysis studies demonstrated the ability ofGVG to attenuate cocaine-induced increases in extracellular DA levels toa similar extent in both areas (Dewey, et al., 1997; Morgan and Dewey,1998).

In the present invention, two different species of rodents and primateswere used to conduct imaging and behavioral experiments. However, themesocorticolimbic DA system is neuroanatomically andneurophysiologically homologous in both species. In addition, thebiochemical effects of cocaine on extracellular DA, measured by in vivomicrodialysis techniques, are similar in both species, and both rodentsand primates readily self-administer cocaine (Morgan, et al., 1998).

Based on the experimental results of the present invention it issubmitted that the blockade of the behaviors in the CPP paradigm was dueto an attenuation of cocaine's effects on brain DA secondary to theGVG-induced increases in GABAergic inhibition of the mesocorticolimbicDA system.

GVG offers the conceptual advantage of blocking cocaine's incentivemotivational and biochemical effects on brain DA by irreversibyinhibiting GABA-T, making the relatively slow de novo synthesis of thisenzyme the rate determining step in reversing the inhibition ofcocaine's effects. A recent case report of a cocaine abuser suggeststhat gabapentin, an anticonvulsant that also potentiates GABAergictransmission via unknown mechanisms, attenuated cocaine withdrawal andcraving. Taken together, these data indicate that drugs selectivelytargeted at the GABAergic system can be beneficial for the treatment ofcocaine addiction. More specifically, GVG-induced GABA-T inhibition,which produces an increase in extracellular brain GABA levels,represents an effective drug and novel strategy for the treatment ofcocaine addiction.

Example 7

The phenomenon of sensitization is observed with virtually all drugs ofaddiction. Sensitization is believed to play a role in the etiology ofaddiction. In this example, the effect of saline and 150 mg/kg i.p. ofGVG on the expression of cocaine-induced stereotypic behavior followinga sensitizing regimen of cocaine was measured in ten freely moving rats.

Animals received 15 mg/kg i.p. of cocaine and stereotypy was determinedin standard locomotor cages. For 6 consecutive days, animals received 15mg/kg i.p. of cocaine once a day in their home cages. Eight days later,animals were rechallenged with 15 mg/kg i.p. of cocaine and stereotypywas determined. A five point rating scale was used to assess stereotypyand the rater was blind to the treatment received by each animal. It wasnoted that GVG abolished the expression of cocaine-induced sensitizationat a dose of 150 mg/kg i.p., when administered 2.5 hours prior to thecocaine challenge. The results are shown in Table XIII below.

TABLE XIII Effect of saline and 150 mg/kg i.p. of GVG on the expressionof cocaine-induced stereotypies following a sensitizing regimen ofcocaine. Sterotypy Treatment 2 hrs. before Stereotypies on Day score onDay 1 measuring Stereotypy Score 15 2.5 ± 0.4 1 ml/kg i.p. of 0.9% NaCl 4.1 ± 0.5* 2.9 ± 0.4 150 mg/kg i.p. of GVG 2.3 ± 0.6 *Significantlygreater than Day 1, p < 0.05, Student's test

The next experiments were designed to determine the effects of GVG onnicotine-induced increases in NACC DA as well as on behaviors associatedwith this bio-chemical effect. Specifically, this was accomplishedby: 1) using in vivo microdialysis in freely moving naive andchronically-nicotine treated animals to measure the effects of GVG andnicotine on extracellular NACC DA; 2) using positron emission tomography(PET) to measure the effect of GVG on nicotine-induced decreases in¹¹C-raclopride binding in the striatum of anesthetized, female baboonsand 3) examining the effect of GVG on nicotine-induced CPP.

Example 8 Effects of GVG on nicotine-induced increases in NACC DA 1.Microdialysis studies in Rodents

In this example, nicotine was used as the addictive drug. In animals of(Group 1), nicotine (0.4 mg/kg, sc) was administered 2.5 hours after GVG(75, 90, 100, or 150 mg/kg, i.p). In a separate series of experiments(Group 2) animals were treated for 21 days with nicotine (0.4 mg/kg,s.c., twice daily). On the day of the study, GVG (100 mg/kg) wasadministered either 2.5, 12 or 24 hours prior to nicotine (0.4 mg/kg,s.c.) challenge. In all studies, animals were placed in themicrodialysis bowls the night before the experiment and artificialcerebrospinal fluid (ACSF) was perfused through the microdialysis probesat a flow rate of 2.0 μl/min. At the end of each study, animals weresacrificed and their brains were removed and sectioned for probeplacement verification.

In Group 1 animals, nicotine increased extracellular DA concentrationsin the NACC by approximately 100%, 80 minutes following administration(FIG. 5A). That is, DA levels were elevated to approximately 200% ofbasal levels. DA returned to basal levels approximately 160 minutesfollowing administration. GVG in a dose-dependent fashion inhibited thisincrease as shown in FIG. 5A. At 75 mg/kg, GVG had no effect onnicotine-induced increases in DA while at 90 mg/kg, GVG inhibited DAincreases by approximately 50% and at 100 mg/kg, it completely abolishedany DA increase. The highest dose of 150 mg/kg completely abolished theeffects as well (data not shown). Of particular note is the finding thatat the three higher doses (90, 100, or 150 mg/kg) GVG lowered basal DAlevels prior to nicotine administration. The lowest dose (75 mg/kg) hadno effect on basal DA levels and subsequently no effect on nicotine'sability to elevate extracellular NACC DA.

In Group 2 animals, nicotine increased extracellular NACC DA levelswithin the same time period and to the same extent measured in Group Ianimals (approximately 100% above baseline, FIG. 5B). Similar to ourfindings in Group 1, when administered 2.5 hours prior to nicotineadministration, GVG (100 mg/kg) completely abolished nicotine-inducedincreases in extracellular DA. However, when administered 12 hours priorto challenge, nicotine increased extracellular DA levels approximately25% above baseline values (FIG. 5B). In Group 2 animals that receivedGVG 24 hours prior to nicotine challenge, extracellular DA levelsincreased to values similar to those measured in control animals (FIG.5B). Consistent with our previous findings (Dewey, et al., 1997), GVGdid not alter gross locomotor activity during the 2.5 hour pretreatmentinterval. However, nicotine increased gross locomotor activity in allanimals regardless of the dose of GVG they received.

Example 9 2. Nicotine-induced CPP in Rodents Description of CPPapparatus

The CPP apparatus was made entirely of plexiglass, except for the floorin one of the pairing chambers, which was made of a stainless steelplate with holes (0.5 mm in diameter) spaced 0.5 mm from edge to edge.The two pairing chambers differed in visual and tactile cues. Onechamber was entirely light blue with the stairless steel floor and thesecond chamber was light blue with horizontal black stripes (2.5 cmwide) spaced 3.8 cm apart with a smooth plexiglass floor. The twopairing chambers were separated by a third, neutral connecting tunnel(10×14×36 cm) with clear plexiglass walls and a plexiglass floor. Thevisual and tactile cues were balanced such that no significant sidepreference was exhibited by animals prior to conditioning.

The effect of GVG on the expression of CPP in Rodents

The conditioning procedure consisted of 20 sessions carried outconsecutively over 20 days. The first three sessions were habituationsessions, during which the animals were handled for 5 minutes per dayand exposed to the sights and sounds of the test room. This was followedby 16 sessions of 8 pairings with 1) vehicle/vehicle (1 ml/kg i.p. 0.9%saline, n=10 animals) or 7 saline-nicotine (0.4 mg/kg s.c.) groups with10 animals in each group. Half the animals in any test group receivednicotine before exposure to the blue chamber and the other half receivesaline before exposure to the blue and black striped chamber. Theanimals that received vehicle or nicotine were injected and confined tothe appropriate compartment for 30 minutes via guillotine plexiglassdoors to block access to the rest of the chamber. The final session (day20) was a test session, in which animals received one of the followingtreatments 30 minutes before the experiment: 1) saline or 2) GVG (18.75,37.5, 75 or 150 mg/kg i.p.). The entrances to both pairing chambers wereopened, and the animals were allowed to freely move between the 3chambers for 15 minutes. The amount of time spent in each chamber wasrecorded using an automated infrared beam electronically coupled to atimer.

The effect of GVG on the acquisition of CPP

The animals were habituated as described above. Animals were giveneither saline or GVG (37.5 and 75 mg/kg i.p.) 2.5 hours before theanimals received nicotine. Subsequently, the animals were then placedinto the appropriate chamber for 30 minutes. This was repeated for 8pairings over a 16 day period. On the test day, animals were placed inthe CPP apparatus and allowed free access to the all of the CPP chambersand the amount of time spent in chamber was recorded.

The administration of saline did not produce a chamber preference.However, nicotine (0.4 mg/kg s.c.) produced a statistically significantand reliable CPP response where animals spent 9.6+0.6 mins on the paired(nicotine) side compared with 5.4+0.6 mins on the unpaired (saline) side(Table XIV and XV). Statistical analysis of the expression dataindicated a treatment effect (F(5, 50)=21.6, p<0.001). Post hoc analysisrevealed that GVG at doses of 18.75, 37.5, 75.0, or 150 mg/kg but notsaline, abolished the expression phase of nicotine-induced CPP (TableXIV).

Analysis of the acquisition data indicated a treatment effect(F(3,32)=11.8, p<0.05). Post hoc analysis indicated that GVG (37.5mg/kg) did not significantly block the acquisition of thenicotine-induced CPP (Table XV). In contrast, at a dose of 75 mg/kg, GVGsignificantly blocked the acquisition phase of nicotine-induced CPP(Table XV).

TABLE XIV Effect of saline and GVG on expression of conditioned placedpreference response to 0.4 mg/kg s.c. of (−) nicotine Time Spent in Druggiven on test chambers (min) Treatment Pairings day Paired UnpairedSaline/Saline Saline² 7.4 ± 0.3¹   7.6 ± 0.3 Saline/Nicotine Saline 9.6± 0.6   5.4 ± 0.6 Saline/Nicotine GVG, 18.75 mg/kg³ 7.5 ± 0.7*   7.5 ±0.7 Saline/Nicotine GVG, 37.5 mg/kg 6.8 ± 1.0**  8.2 ± 1.0Saline/Nicotine GVG, 75 mg/kg 6.4 ± 0.3**  8.6 ± 0.3 Saline/NicotineGVG, 150 mg/kg 5.0 ± 0.9** 10.0 ± 0.9 ¹Each value represents the meannumber of minutes spent in each chamber ± S.E.M. A total of 8-10 ratswere examined for each treatment pairing. All animals received 8pairings with nicotine and saline prior to the test day. On the testday, animals received either saline or GVG 2.5 hours before being placedinto the CPP apparatus. ²Saline was 1 ml/kg s.c. of 0.9% saline.*Significantly less than Saline/Nicotine pairing with saline on testday, P < 0.05, ANOVA and Student-newman-Keuls test. **Significantly lessthan Saline/Nicotine pairing with saline on test day, P < 0.01, ANOVAand Student-Newman-Keuls test.

TABLE XV Effect of saline and GVG on acquisition of conditioned placepreference response to 0.4 mg/kg s.c. of (−)-nicotine Time spent inchambers (min) Treatment Pairings Paired Unpaired Saline/Saline² 7.3 ±0.3¹ 7.7 ± 0.3 Saline/Nicotine 9.6 ± 0.6¹ 5.4 ± 0.6 Nicotine/GVG, 37.5mg/kg 8.8 ± 0.5  6.2 ± 0.5 i.p. Nicotine/GVG, 75 mg/kg 6.9 ± 0.9* 8.1 ±0.9 i.p. ¹Each value represents the mean number of minutes spent in eachchamber ± S.E.M. A total of 8-10 rats were examined for each treatmentpairing. Animals were pretreated with either saline, 37.5 or 75 mg/kgi.p. of GVG and 2.5 hours later, each animal received 0.4 mg/kg s.c. ofnicotine, except for one group, which received saline followed by salinetreatment (saline/saline pairing). Eight pairings were performed witheach animal. ²The saline was 1 ml/kg s.c. of 0.9% saline. *Significantlyless than Saline/Nicotine pairing with saline on test day, P < 0.05,ANOVA and Student-Newman-Keuls test.

Primate PET studies

Adult female baboons (n=16) (Papio anubis, 13-18 kg) were used for allimaging studies and carbon-11 labeled raclopride (¹¹C-raclopride).Animals were placed into 5 groups as detailed in Table XVI. Controlanimals (Group 1) received two injections of ¹¹C- raclopride without anydrug intervention in order to determine the test/retest variability ofthe measurement. These data have been reported previously (Dewey et al.,1998). Group 2 animals received GVG alone (300 mg/kg) 2.5 hours prior tothe second injection of ¹¹C-raclopride. Like Group 1 animals, these datahave been reported previously (Dewey, et al., 1992). Group 3 animalsreceived nicotine alone (0.3 mg total, approximately 0.02 mg/kg) 30minutes prior to the second injection of ¹¹C-raclopride. In the combinedGVG/nicotine studies, GVG was administered intravenously (i.v.) at dosesof 100 (Group 4) or 300 mg/kg (Group 5) 2.5 hours prior to nicotineadministration. Nicotine (0.3 mg total, i.v.) was administered 30minutes prior to the second injection of ¹¹C-raclopride. Arterial bloodsamples were obtained throughout the study and selected plasma sampleswere analyzed for the presence of unchanged ¹¹C-raclopride. Animals werenot removed from the gantry between isotope injections. Data analysiswas performed using the Logan method as detailed previously (Logan, etal., 1990).

Each primate (n=16) received two ¹¹C-raclopride injections. The firstserved as a baseline for the second that followed GVG, nicotine, orboth. Test/retest primates (n=7, Group 1, Table XVI) received placebo(0.9% saline, 1 ml/kg) 30 mins prior to the second radiotracer injectionin order to determine the test/retest variability of the method. Allremaining primates (n=9) received a systemic injection of GVG, nicotineor both prior to the second [¹¹C]-raclopride injection.

As reported previously (Dewey, et al., 1998), the test/retest meandistribution volume (DV) ratio variability of labeled raclopride in theprimate striatum was slightly greater that 7% (Table XVII). GVGadministration (300 mg/kg, Group 2) significantly increased the mean DVratio by 18% (Table XVII). These data are consistent with microdialysisstudies demonstrating that GVG dose dependently decreases extracellularDA in freely moving animals. Nicotine administration (Group 3), however,produced the opposite effect of GVG and significantly reduced the meanDV ratio by 12% (Table XVII). This is again consistent with ourmicrodialysis data demonstrating that nicotine increases extracellularDA in freely moving animals. When administered sequentially, GVG (100mg/kg, Group 4) abolished the decrease in the mean DV ratio produced bynicotine alone (Group 3). At this dose of GVG, the mean DV ratio wassimilar to the test/retest value obtained in Group 1 animals (9%, TableXVII). However, when administered at a dose of 300 mg/kg (Group 5), themean DV ratio for labeled raclopride was significantly higher (15%) thanthe test/retest values and was in fact, similar to the values obtainedin Group 2 animals that received GVG alone (Table XVII).

It was noted that GVG, nicotine or both did not alter the rate ofsystemic metabolism of labeled raclopride nor the regional distributionof the radiotracer. Recovery from each study was unremarkable.

TABLE XVI Groups for Primate PET Studies Group Condition 1 Test/Retest(no challenge) 2 GVG (300 mg/kg) 3 Nicotine (0.3 mg) 4 GVG (100 mg/kg),Nicotine (0.3 mg) 5 GVG (300 mg/kg), Nicotine (0.3 mg)

TABLE XVII Effects of Drug Challenge on the Mean DV Ratio Group % Changein Mean DV Ratio 1 7.16 ± 1.2 2 18.8 ± 3.2 3 −12.3 ± 2.6   4 9.45 ± 2.15 15.1 ± 2.8

Discussion of Experimental Results Obtained in Example 9

In this example, we demonstrated that nicotine (0.4 mg/kg s.c.)increased NACC DA by approximately 100% (or 200% above baseline) infreely moving animals approximately 80 minutes following administration.Previous microdialysis studies have reported that nicotineadministration at doses of 0.6 or 0.8 mg/kg (s.c.) produced a 220% and179% increase in extracellular DA levels in the NACC, respectively, (DiChiara and Imperato, 1988; Imperato et al., 1986; Brazell et al., 1990).Although not directly comparable, our results are clearly in line withthese earlier findings. Furthermore, in our animals exposed chronicallyto nicotine, a nicotine challenge produced a 90% increase inextracellular NACC DA levels. This finding is consistent with previousdata indicating that chronic nicotine administration does not producetolerance or sensitization to an acute challenge with nicotine (Damsmaet al., 1989).

with respect to our findings using GVG, we demonstrated that itdose-dependently inhibited nicotine-induced increases in NACC DA in bothnaïve and chronically nicotine treated animals. This is the first studyto report such an action of GVG. At a dose of 75 mg/kg, GVG had noeffect as nicotine increased extracellular DA by nearly 200% while adose of 90 mg/kg produced an inhibition of nearly 50%. At the twohighest doses examined (100 and 150 mg/kg) GVG completely abolishednicotine-induced increases in extracellular NACC DA levels. Previously,we demonstrated that an acute injection of GVG (300 mg/kg i.p) produceda 25% decrease in cocaine induced increases in NACC DA (Dewey et al.,1998). However, chronic treatment with GVG, at a similar dose, produceda greater inhibition (Morgan and Dewey, 1998). Together these data showthat the dose of GVG needed to significantly attenuate drug-inducedincreases in NACC DA levels is dependent not only on the challenge drugused (e.g., cocaine, nicotine), but also on the dose at which thechallenge drug is administered.

The present data further demonstrates that the effectiveness of GVG isrelated to its dose dependent ability to lower basal DA concentrationsprior to drug challenge. For example, the 75 mg/kg dose had no effect onbasal DA and on nicotine-induced increases in DA. However, at a dose ofeither 90 or 100 mg/kg, GVG lowered basal DA levels and reduced by 50%or abolished the effects of nicotine, respectively. Therefore, itappears that the dose-dependent attenuation of either nicotine orcocaine-induced increases in NACC DA is due to a pre-lowering of basalDA concentrations, subsequent to an increase in endogenous GABA producedby GVG. This is consistent with data indicating that augmentation ofGABAergic function reduces DA in the NACC.

In an extension of our previous work with GVG and cocaine, we examinedthe temporal course of GVG's effects on nicotine-induced increases inNACC DA in animals chronically treated with nicotine for 21 days. Whenadministered 2.5 hours prior to nicotine at a dose of 100 mg/kg, GVGcompletely abolished drug-induced increases in NACC DA. However, whenadministered at the same dose 12 hours prior to challenge, nicotineincreased extracellular DA by approximately 25%.

GVG had no effect on nicotine-induced increases in NACC DA, when it wasadministered 24 hours prior to nicotine challenge at the same dose.Clearly, our microdialysis and behavioral data show that even smallchanges in GABA-T inhibition produced by increasing doses of GVG have aprofound effect on the inhibition of nicotine-induced elevations in NACCDA and CPP, respectively.

These data are particularly interesting in light of the synthesis rateof GABA-T, the half-life of GVG in the rodent brain, the duration of theeffect on GABA, and the sharp dose response curve detailed here.Previous findings demonstrate that the biologic half-life of GABA-T inthe rodent brain is 3.4 days while the half-life of GVG in the brain isapproximately 16 hours. In addition, total brain GABA levels do notbegin to decrease until 24 hours following acute GVG administration(Jung, et al., 1977). The disparity between the sustained brain GABAlevels measured 24 hours following a single dose of GVG and the normalresponse to a nicotine challenge observed at the same time pointsuggests that GABAergic inhibition of the mesotelencephalic rewardpathway may not be a simple reflection of total brain GABA levels. Thatis, while total brain GABA levels are still significantly elevated 24hours following an acute dose of GVG, small functional differences inspecific pathways may be masked by these global measurements. Finally,it is conceivable that GABA receptors have become desensitized to GABAover the 24 hour period, however, we are unaware of any evidence in theGABA system that would support such an hypothesis.

In the present study, we demonstrated that 8 saline-nicotine pairingsproduced a reliable CPP response. Our results are in agreement withprevious studies indicating that nicotine (0.1-1.2 mg/kg s.c.) producesa dose-dependence in the CPP response of male Sprague-Dawley animals(Fudala et al., 1985; Fudala and Iwamoto, 1986). We have also shown thatLewis, but not F344 animals, show a CPP response to nicotine after 10pairings (Horan et al., 1997). However, a previous report has shown that4 nicotine-vehicle pairings did not elicit a CPP response in male hoodedanimals (Clarke and Fibiger, 1987). Thus, it appears thatnicotine-induced CPP may be species dependent, although this may beconfounded by the fact that the studies quoted utilized a differentnumber of pairings. The nicotine-induced CPP response reported in thepresent study is consistent with the notion that nicotine produces apositive effect on incentive motivational behavior.

This data, for the first time, demonstrates that GVG can block thebiochemical and behavioral effects of nicotine using the CPP paradigm.The CPP data clearly indicate that at a dose as low as 18.75 mg/kg, GVGabolishes the expression of the CPP response produced by nicotine. Ourdata also indicated that a dose of 75 mg/kg, but not 37.5 mg/kg, blockedthe acquisition of the CPP response to nicotine. Based on these dosefindings, the dose of GVG needed for the treatment of smoking cessationcan be a total of 250-500 mg a day (compared with 2-4 grams/day forepilepsy), a range considerably lower than that given to epileptics.

The effects of GVG on nicotine-induced CPP are unlikely to be related toits producing a rewarding or aversive effect as we have previously shownthat GVG alone (75-300 mg/kg i.p.) does not produce CPP or aversion(Dewey et al., 1998). Furthermore, it is unlikely that GVG abolishesnicotine's behavioral actions by interfering with memory or locomotoractivity as GVG does not block food reward or locomotor activity atdoses as high as 300 mg/kg (Dewey et al., 1998).

Finally, it has been shown that GVG is not self-administered by rhesusmonkeys and animals withdrawn from chronic GVG treatment do not exhibitwithdrawal signs or symptoms (Takada, and Yanagita, 1997). Thus, GVG,unlike other drugs used in the pharmacotherapy of certain addictions(e.g. methadone, antabuse), is itself not addicting and does not producesignificant aversive effects.

The attenuation of the acquisition of the CPP response to nicotine byGVG can be interpreted as a decrease in the positive incentive value ofnicotine. These data show that GVG decreases the likelihood that ananimal will acquire the association of a positive incentive effectfollowing nicotine administration. Interestingly, our results indicatedthat the dose of GVG required to block the expression phase of the CPPresponse produced by nicotine was ¼ of the amount needed to block theacquisition of the CPP response. This finding is congruent with ourprevious data indicating that a higher dose of GVG was required to blockthe acquisition, as opposed to the expression of CPP to cocaine (Deweyet al., 1998). The explanation for this difference is unknown. Since GVGattenuates the expression of the CPP response to nicotine, thisdemonstrates that GVG is decreasing the drug-seeking behavior of theanimal as the animal has already acquired the positive incentive valueof the drug.

Thus, our data shows that GVG can be more effective in blocking thecraving for nicotine than it is at blocking the positive incentive valueor rewarding action of nicotine. Finally, at the highest dose tested,150 mg/kg, GVG produced a significant aversive response on the test day(Table XV) where animals spent 5.0+0.9 on the paired (nicotine) side and10.0+0.9 minutes on the unpaired (saline) side. These data suggest thatthere might be a ceiling effect at which GVG in high doses becomesaversive in animals treated with nicotine and tested in a drug-freestate. These data may have implications in developing the dose limits tobe tested in human clinical trials.

Based on our knowledge of the CPP paradigm, our data support thefollowing results. In the CPP paradigm, animals are tested, in adrug-free state, to determine whether they prefer an environment inwhich they previously received nicotine as compared to an environment inwhich they previously received saline. If the animal, in a drug-freestate, consistently chooses the environment previously associated withnicotine, the inference is drawn that the appetitive value of nicotinewas encoded in the brain and is accessible in the drug-free state(Gardner, 1997). Indeed, on the test day, the approach and associationof the animals with the drug-paired side can be considered drug-seekingbehavior. In essence, environmental stimuli and other cues that werepreviously neutral or lacked salience have through repeated pairingswith nicotine, become salient. Subsequently, when the animals arere-exposed to these cues, a CPP response is produced, i.e. the cues canelicit the drug effect. Thus, drug-related cues produce a Pavlovianconditioned response.

This is critical as it is known that non-pharmacologic factors, inaddition to pharmacologic ones, play a role in mediating the incentivevalue of drugs of addiction (Jarvik and Henningfield, 1988). In fact, ithas been demonstrated clinically that in detoxified addicts, exposure tostimuli that were previously associated with drug use, can elicitrelapse (Childress et al., 1986a,b; Childress et al., 1988; Ehrman etal., 1992; O'Brien et al., 1992; Wikler, 1965). Thus, these data showthat since GVG blocks the expression of the nicotine-induced CPPresponse, then GVG blocks the craving or seeking of nicotine. Therefore,GVG is effective in the treatment of individuals who have the desire tostop smoking cigarettes. These data further show that GVG is effectivein abolishing the expression of the CPP response to nicotine and canattenuate craving in the face of environmental cues previouslyassociated with smoking.

Our primate PET data are consistent with previous findings usingmultiple pharmacologic challenges that demonstrate ¹¹C-raclopridebinding is sensitive to both increases and decreases in synaptic DA(Dewey, et al., 1993; Seeman, et al., 1989).

As evidenced in Group 3 animals ( Table XVII), the mean DV ratio wasconsistently decreased relative to baseline values following nicotineadministration. This decrease exceeded the test/retest variability oflabeled raclopride and is less than the decrease measured with GBR-12909(Dewey, et al., 1993) or scopolamine (Dewey, et al., 1993). Pretreatmentwith GVG at a dose of 100 mg/kg 2.5 hours prior to nicotine produced amean DV ratio similar to Group 1 animals (Table XVII). However, when thedose of GVG was increased to 300 mg/kg, the mean DV ratio was elevatedto values consistent with Group 2 animals. These data show that thelower dose of GVG produced a decrease in synaptic DA roughly equivalentto the increase produced by nicotine while the higher dose of GVGproduced a decrease that far exceeded nicotine's ability to increase DA.Our microdialysis studies support these data that higher doses of GVGproduce a greater decrease in extracellular DA in freely moving animals.

The microdialysis and PET findings combined with the CPP data show thatincreases in DA in the NACC alone underlie the addictive liability ofdrugs of abuse. First, these data, combined with the above data forcocaine, show that in vivo microdialysis studies or PET measurements ofendogenous DA alone is not necessarily predicative of the efficacy ofdrugs used to treat diseases thought to be neurotransmitter-specific innature. Second, both the microdialysis data and the PET data clearlydemonstrate that at a dose of 100 mg/kg, GVG completely blockednicotine-induced increases in NACC DA levels, whereas a dose of 75 mg/kghad no effect. In contrast, GVG, at a dose as low as 18.75 mg/kg,completely abolished the expression phase of nicotine-induced CPP whileit took a dose of 75 mg/kg to abolish the acquisition phase.

Based upon the dose-response curve obtained from the microdialysis data,GVG at a dose of 18.75 mg/kg would not be expected to have any effect onnicotine-induced increases in NACC DA. Furthermore, a similar effect wasnoted using cocaine where a dose of 300 mg/kg of GVG reducedcocaine-induced increases in NACC DA levels by 25%, while a dose of 150mg/kg completely abolished the expression and acquisition phase ofcocaine-induced CPP (Dewey, et al., 1997; 1998).

Together, these data suggest at least two plausible and perhaps combinedexplanations. First, differential changes in DA following pharmacologicchallenge in regions other than the NACC alone may be responsible forthe addictive liability of a particular drug. Indeed, it has beenreported that various addictive drugs can alter DA levels in brain areasother than the NACC including the amygdala, corpus striatum, and frontalcortex, (Hurd, et al., 1997; Dewey, et al., 1997; Di Chiara andImperato, 1988; Marshall, et al., 1997). Second, neurotransmitters otherthan DA may play a vital role in the addictive liability of drugs ofabuse. For example, a CPP response to cocaine is still maintained inmice that lack the DA and 5-HT transporters (Sora, et al., 1998; Rocha,et al., 1998). Furthermore, it is known that neurotransmitters such as5-MT, acetylcholine, enkephalins and glutamate, play a role in mediatingthe effects of addictive drugs, including nicotine (Bardo, 1998;Gardner, 1997). Taken together, these data show that GVG inhibits theeffects of cocaine and nicotine through changes in DA in regions otherthan the NACC. Concomitantly, GVG may be inhibiting otherneurotransmitters that either modulate DA directly or are themselvesinvolved in mediating the effects of drugs of addiction. Further studiesdesigned to assess the multiple effects of GVG on otherneurotransmitters are ongoing.

Previously, we demonstrated that the ability of GVG to attenuatecocaine-induced increases in NACC DA is completely abolished bypretreating animals with the selective GABAB receptor antagonist SCH50911 (Bolser et al., 1995), a drug that does not significantly alter DAlevels when given alone. Therefore, it can be shown that GVG abolishesthe action of nicotine via its increase in GABA levels, whichsubsequently stimulates GABAB receptors. This is consistent with dataindicating that the administration of baclofen, a selective GABABagonist (Bowery and Pratt, 1992; Kerr et al., 1990), into the VTAsignificantly attenuates the CPP response in animals produced bysystemic morphine (Tsuji et al., 1995). Furthermore, systemicadministration of baclofen attenuates cocaine self-administration on aprogressive ratio and discrete trials schedule (Roberts et al., 1996,1997).

It can be argued that GVG attenuates the pharmacologic and behavioralactions of nicotine simply by altering the amount that effectivelyenters the brain either by changing blood brain barrier permeability orby increasing the systemic rate of metabolism of nicotine. Thispossibility is unlikely for a number of reasons. First, GVG had noeffect on the blood brain barrier transport of ¹¹C-cocaine, an alkaloidpreviously shown to increase NACC DA, in both the rodent or primatebrain. Second, GVG is excreted primarily in the unchanged form by thekidneys (Grant and Heel, 1991; Porter and Meldrum, 1998), whereasnicotine is metabolized by enzymes in the liver. Finally, GVG does notinteract with the hepatic microsomal enzymes (Grant and Heel, 1991;Porter and Meldrum, 1998) and thus would not induce or inhibit theseenzymes.

The size of the NACC is well below the resolution of our tomographmaking its specific analysis outside the capabilities of this technique.Therefore, our analysis included the corpus striatum, bilaterally andthe cerebellum. Marshall et al. (1995) have demonstrated that nicotineincreased DA equally in both the NACC and the corpus striatum, while ourown microdialysis data demonstrates that GVG decreases DA concentrationsequally in both regions as well (Dewey, et al., 1997). These primatedata further support the use of this imaging technique to evaluate thefunctional consequences of pharmacologic challenges in the intact livingbrain.

Furthermore, this medical imaging technique provides a unique windowinto the interactions that have been shown to exist betweenfunctionally-linked neurotransmitters in both the primate and humanbrain.

Combined with an exhaustive literature supporting the fundamentalprinciple that neurotransmitters interact in both functionally-specificand regionally specific neuroanatomic foci, it is becoming increasinglyclear that new treatment strategies for brain disorders (includingaddictions to cocaine, nicotine, heroin, methamphetamine and alcohol)can be implemented with a more global awareness of this fundamental andwell-documented principle. While changes in individual neurotransmitterconcentrations may indeed underlie the etiology of a specific disorder,it is likely that disease progression and symptom development are linkedto compensatory or disease-induced changes in other neurotransmittersfunctionally-linked to the original target. With this knowledge, we havedeveloped novel treatment strategies specifically designed to alter oneor more neurotransmitters by targeting another. Our findings withnicotine, cocaine, methamphetamine, alcohol and GVG represent greatutility for treatment of mammals addicted to drugs of abuse.

Example 10 Effects of GVG on methamphetamine-induced increases in NACCDA

In this example, the effects of GVG on methamphetamine-induced changeson NACC dopamine concentrations was studied in 6-8 freely moving rats.Methamphetamine at a dose of 1.25 mg/kg i.p. and 2.5 mg/kg i.p. wasadministered to the animals. It was noted that methamphetamine elevatedextracellular DA concentrations in the NACC by approximately 2500% overbasal levels, 100 minutes following administration of 2.5 mg/kg andapproximately 1500% over basal levels following administration of 1.25mg/kg (FIG. 6). DA returned to basal levels approximately 200 minutesfollowing administration.

When GVG was administered prior to methamphetamine administration, GVGdose-dependently inhibited the DA increase as shown in FIG. 7. At 300mg/kg, GVG inhibited increases in DA by approximately 38% and at 600mg/kg it inhibited increases in DA by approximately 58%. These datademonstrate that GVG inhibits methamphetamine increases in extracellulardopamine concentrations in the NACC.

Thus, it is noted from the above data that the rank order of nicotine,cocaine and methamphetamine to increase NACC DA levels ismethamphetamine (2500%)>cocaine (450%)>nicotine (90%) which parallelsthe rank order of the size of an acute dose of GVG needed tosignificantly decrease drug-induced increases in NACC DA.

Example 11 Effects of GVG on ethanol-induced increases in NACC DA

In this example, the effects of GVG on ethanol-induced changes on NACCdopamine concentrations was studied in 6-8 freely moving rats. Ethanolat a dose of 1.0 g/kg i.p. was administered to the animals. Ethanolincreased elevated extracellular DA concentrations in the NACC byapproximately 200% over basal levels at approximately 125 minutesfollowing ethanol administration.

When GVG was administered at a dose of 300 mg/kg, it inhibited increasesin DA by approximately 50% (FIG. 8). Also, at a dose of 100 mg/kg, GVGsignificantly inhibits, by approximately 40%, alcohol's ability toincrease nucleus accumbens dopamine in freely moving rats (data notshown). These data demonstrate that GVG inhibits ethanol increases inextracellular dopamine concentrations in the NACC.

Example 12 Effects of GVG on cocaine/heroin induced increases in NACC DA

In this example, we investigated the effects of GVG on the synergisticelevations in NAc DA following a cocaine/heroin (speedball) challenge.In vivo microdialysis studies were performed using adult maleSprague-Dawley rats (Taconic Farms) as detailed previously (Morgan andDewey, 1998). Cocaine, a dopamine reuptake inhibitor, (n=6-8) wasadministered (i.p.) at a dose of 20 mg/kg while heroin, an indirectdopamine releaser, (n=6-8) was administered (i.p.) at a dose of 0.5mg/kg. In studies designed to investigate the synergistic effects of acocaine/heroin combination (n=6-8), both drugs were administered at theidentical dose used in the single drug studies. Alone cocaine produced amarked elevation in extracellular DA of approximately 380% abovebaseline values, 60 minutes following administration. DA returned tobaseline within 120 minutes. In contrast, heroin increased NAc DA byonly 70%, 60 minutes following administration, returning to baselinewithin 140 minutes. However, when combined, the two drugs produced anincrease in NAc DA of approximately 1000%, 180 minutes followingadministration that had not returned to baseline values by 200 minutesafter reaching peak values (FIG. 9). This increase was significantlydifferent (P>0.001) from cocaine or heroin alone.

This neurochemical synergy, as compared to an additive effect, wasevident not only in the magnitude of the increase in NAc DA, but also inthe time it took to reach the peak elevation and return to baselinevalues. Individually, each drug produced a maximum increase within 60minutes following challenge. When combined, however, this maximumincrease took nearly three times longer to achieve than either drugalone. Furthermore, it took considerably longer to return to baselinevalues when compared to each drug separately. These findings are showthat the duration of the euphoria is much longer when both drugs areused in combination as opposed to separately.

With respect to the absolute magnitude of the response, GVG completelyabolished the synergistic effects following the combined drug challenge.In animals that received GVG (300 mg/kg, I. p.) 2.5 hours prior tochallenge, NAc DA increased by approximately 500% 180 minutes followingchallenge (FIG. 9). This increase was significantly different from bothcocaine and heroin alone (P>0.05 and 0.001, respectively) andcocaine/heroin combined (P>0.001). The data obtained followingpretreatment with GVG is similar to an additive effect of both cocaine(380%) and heroin (70%) compared to a synergistic effect.

While abolishing the synergistic effect of both drugs on the absolutemagnitude of the increase, GVG did not effect the temporal aspects ofthe response. Following GVG administration and a subsequentcocaine/heroin challenge, NAc DA reached a maximum concentration within180 minutes which is identical to the response measured in animals thatdid not receive GVG prior to challenge.

The results of this example show that GVG effectively attenuates thesynergistic elevations in NAc DA produced by a cocaine/heroin challenge.Combined with our previous studies, this finding show the effectivenessof GVG for the treatment of poly-drug abuse.

The above examples demonstrate that drugs that selectively target theGABAergic system can be beneficial for the treatment of drugs of abuse,such as psychostimulants, narcotic analgesics, alcohols and nicotine orcombinations thereof. More specifically, GVG-induced GABA-T inhibition,which produces an increase in extracellular brain GABA levels,represents an effective drug and novel strategy for the treatment ofcocaine, nicotine, heroin, methamphetamine and ethanol addiction.

Thus, while there have been described what are presently believed to bethe preferred embodiments of the present invention, those skilled in theart will realize that other and further embodiments can be made withoutdeparting from the spirit of the invention, and it is intended toinclude all such further modifications and changes as come within thetrue scope of the claims set forth herein.

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What is claimed is:
 1. A method for changing addiction-related behaviorof a mammal suffering from addiction to any combination of abused drugsselected from the group consisting of psychostimulant, narcoticanalgesics, alcohols, and nicotine which comprises administering to themammal an effective amount of gamma vinylGABA (GVG) or apharmaceutically acceptable salt thereof, or an enantiomer or a racemicmixture thereof, wherein the effective amount is sufficient to diminish,inhibit or eliminate behavior associated with craving or use of saidcombination of abused drugs.
 2. A method of claim 1, wherein saidelimination of behavior associated with craving said combination ofabused drugs occurs in the absence of an aversive response or appetitiveresponse to GVG.
 3. A method of claim 1, wherein said combination ofabused drugs is a combination of drugs selected from the groupconsisting of cocaine, nicotine, methamphetamine, ethanol, morphine andheroin.
 4. A method of claim 1, wherein said combination is cocaine andheroin.
 5. A method of claim 1, wherein GVG is administered in an amountof about 15 mg/kg to about 600 mg/kg.
 6. A method of claim 1, whereinsaid addiction related behavior is conditioned place preference.
 7. Amethod for changing addiction-related behavior of a mammal sufferingfrom addiction to any combination abused drugs selected from the groupconsisting of psychostimulants, narcotic analgesics, alcohols andnicotine which comprises administering to the mammal an effective amountof gamma vinylGABA (GVG) or a pharmaceutically acceptable salt thereof,or an enantiomer or a racemic mixture thereof, wherein the effectiveamount attenuates the rewarding/incentive effect of said combination ofabused drugs in the absence of altering rewarding/incentive effects offood in said mammal.
 8. A method of claim 7, wherein said combination ofabused drugs is a combination of drugs selected from the groupconsisting of cocaine, nicotine, methamphetamine, ethanol, morphine andheroin.
 9. A method of claim 7, wherein said combination is cocaine andheroin.
 10. A method of claim 7, wherein the rewarding/incentive effectsof the combination of abused drugs is attenuated in the absence of analteration in the locomotor function of said mammal.
 11. A method ofameliorating effects of addiction to any combination of abused drugsselected from the group consisting of psychostimulant, narcoticanalgesic, alcohol and nicotine which comprises administering to amammal an effective amount of gamma vinylGABA (GVG) or apharmaceutically acceptable salt thereof, or an enantiomer or a racemicmixture thereof, wherein the effective amount is sufficient to reducedependency characteristics of said combination of abused drugs.
 12. Themethod of claim 11, wherein said combination of abused drugs is selectedfrom the group consisting of cocaine, nicotine, methamphetamine,ethanol, morphine and heroin.
 13. A method of claim 11, wherein saidcombination is cocaine and heroin.
 14. A method of claim 11, wherein GVGis administered in an amount from about 15 mg/kg to about 600 mg/kg. 15.A method of claim 11, wherein said dependency characteristics arereduced in the absence of an aversive response or appetitive response toGVG.
 16. A method of claim 11, wherein said dependency characteristicsare reduced in the absence of an alteration in the locomotor function ofsaid mammal.